Therapeutic compositions

ABSTRACT

This application relates to therapeutic siRNA agents and methods of making and using the agents.

RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No. 10/548,611, filed Aug. 22, 2006, which is the National Stage of International Application No. PCT/US2004/007070, filed Mar. 8, 2004, which claims the benefit of Application No. 60/452,682, filed Mar. 7, 2003; Application No. 60/462,894, filed Apr. 14, 2003; and Application No. 60/465,665, filed Apr. 25, 2003; Application No. 60/463,772, filed Apr. 17, 2003; Application No. 60/465,802, filed Apr. 25, 2003; Application No. 60/493,986, filed Aug. 8, 2003; Application No. 60/494,597, filed Aug. 11, 2003; Application No. 60/506,341, filed Sep. 26, 2003; Application No. 60/518,453, filed Nov. 7, 2003; Application No. 60/454,265, filed Mar. 12, 2003; Application No. 60/454,962, filed Mar. 13, 2003; Application No. 60/455,050, filed Mar. 13, 2003; Application No. 60/469,612, filed May 9, 2003; Application No. 60/510,246, filed Oct. 9, 2003; Application No. 60/510,318, filed Oct. 10, 2003. The contents of the above applications are hereby incorporated by reference in their entirety.

TECHNICAL FIELD

The invention relates to RNAi and related methods, e.g., methods of making and using iRNA agents.

BACKGROUND

RNA interference or “RNAi” is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNA (dsRNA) can block gene expression when it is introduced into worms (Fire et al. (1998) Nature 391, 806-811). Short dsRNA directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. RNAi may involve mRNA degradation.

SUMMARY

A number of advances related to the application of RNAi to the treatment of subjects are disclosed herein. For example, the invention features iRNA agents targeted to specific genes; palindromic iRNA agents; iRNA agents having non canonical monomer pairings; iRNA agents having particular structures or architectures e.g., the Z—X—Y or asymmetrical iRNA agents described herein; drug delivery conjugates for the delivery of iRNA agents; amphipathic substances for the delivery of iRNA agents, as well as iRNA agents having chemical modifications for optimizing a property of the iRNA agent. The invention features each of these advances broadly as well as in combinations. For example, an iRNA agent targeted to a specific gene can also include one or more of a palindrome, non canonical, Z—X—Y, or asymmetric structure. Other nonlimiting examples of combinations include an asymmetric structure combined with a chemical modification, or formulations or methods or routes of delivery combined with, e.g., chemical modifications or architectures described herein. The iRNA agents of the invention can include any one of these advances, or pairwise or higher order combinations of the separate advances.

In one aspect, the invention features iRNA agents that can target more than one RNA region, and methods of using and making the iRNA agents.

In another aspect, an iRNA agent includes a first and second sequence that are sufficiently complementary to each other to hybridize. The first sequence can be complementary to a first target RNA region and the second sequence can be complementary to a second target RNA region.

In one embodiment, the first and second sequences of the iRNA agent are on different RNA strands, and the mismatch between the first and second sequences is less than 50%, 40%, 30%, 20%, 10%, 5%, or 1%.

In another embodiment, the first and second sequences of the iRNA agent are on the same RNA strand, and in a related embodiment more than 50%, 60%, 70%, 80%, 90%, 95%, or 1% of the iRNA agent is in bimolecular form.

In another embodiment, the first and second sequences of the iRNA agent are fully complementary to each other.

In one embodiment, the first target RNA region is encoded by a first gene and the second target RNA region is encoded by a second gene, and in another embodiment, the first and second target RNA regions are different regions of an RNA from a single gene. In another embodiment, the first and second sequences differ by at least 1 and no more than 6 nucleotides.

In certain embodiments, the first and second target RNA regions are on transcripts encoded by first and second sequence variants, e.g., first and second alleles, of a gene. The sequence variants can be mutations, or polymorphisms, for example.

In certain embodiments, the first target RNA region includes a nucleotide substitution, insertion, or deletion relative to the second target RNA region.

In other embodiments, the second target RNA region is a mutant or variant of the first target RNA region.

In certain embodiments, the first and second target RNA regions comprise viral, e.g., HCV, or human RNA regions. The first and second target RNA regions can also be on variant transcripts of an oncogene or include different mutations of a tumor suppressor gene transcript. In one embodiment, the oncogene, or tumor suppressor gene is expressed in the liver. In addition, the first and second target RNA regions correspond to hot-spots for genetic variation.

In another aspect, the invention features a mixture of varied iRNA agent molecules, including one iRNA agent that includes a first sequence and a second sequence sufficiently complementary to each other to hybridize, and where the first sequence is complementary to a first target RNA region and the second sequence is complementary to a second target RNA region. The mixture also includes at least one additional iRNA agent variety that includes a third sequence and a fourth sequence sufficiently complementary to each other to hybridize, and where the third sequence is complementary to a third target RNA region and the fourth sequence is complementary to a fourth target RNA region. In addition, the first or second sequence is sufficiently complementary to the third or fourth sequence to be capable of hybridizing to each other. In one embodiment, at least one, two, three or all four of the target RNA regions are expressed in the liver. Exemplary RNAs are transcribed from the apoB-100 gene, glucose-6-phosphatase gene, beta catenin gene, or an HCV gene.

In certain embodiments, the first and second sequences are on the same or different RNA strands, and the third and fourth sequences are on same or different RNA strands.

In one embodiment, the mixture further includes a third iRNA agent that is composed of the first or second sequence and the third or fourth sequence.

In one embodiment, the first sequence is identical to at least one of the second, third and fourth sequences, and in another embodiment, the first region differs by at least 1 but no more than 6 nucleotides from at least one of the second, third and fourth regions.

In certain embodiments, the first target RNA region comprises a nucleotide substitution, insertion, or deletion relative to the second, third or fourth target RNA region.

The target RNA regions can be variant sequences of a viral or human RNA, and in certain embodiments, at least two of the target RNA regions can be on variant transcripts of an oncogene or tumor suppressor gene. In one embodiment, the oncogene or tumor suppressor gene is expressed in the liver.

In certain embodiments, at least two of the target RNA regions correspond to hot-spots for genetic variation.

In one embodiment, the iRNA agents of the invention are formulated for pharmaceutical use. In one aspect, the invention provides a container (e.g., a vial, syringe, nebulizer, etc) to hold the iRNA agents described herein.

Another aspect of the invention features a method of making an iRNA agent. The method includes constructing an iRNA agent that has a first sequence complementary to a first target RNA region, and a second sequence complementary to a second target RNA region. The first and second target RNA regions have been identified as being sufficiently complementary to each other to be capable of hybridizing. In one embodiment, the first and second target RNA regions are on transcripts expressed in the liver.

In certain embodiments, the first and second target RNA regions can correspond to two different regions encoded by one gene, or to regions encoded by two different genes.

Another aspect of the invention features a method of making an iRNA agent composition. The method includes obtaining or providing information about a region of an RNA of a target gene (e.g., a viral or human gene, or an oncogene or tumor suppressor, e.g., p53), where the region has high variability or mutational frequency (e.g., in humans). In addition, information about a plurality of RNA targets within the region is obtained or provided, where each RNA target corresponds to a different variant or mutant of the gene (e.g., a region including the codon encoding p53 248Q and/or p53 249S). The iRNA agent is constructed such that a first sequence is complementary to a first of the plurality of variant RNA targets (e.g., encoding 249Q) and a second sequence is complementary to a second of the plurality of variant RNA targets (e.g., encoding 249S). The first and second sequences are sufficiently complementary to hybridize. In certain embodiments, the target gene can be a viral or human gene expressed in the liver.

In one embodiment, sequence analysis, e.g., to identify common mutants in the target gene, is used to identify a region of the target gene that has high variability or mutational frequency. For example, sequence analysis can be used to identify regions of apoB-100 or beta catenin that have high variability or mutational frequency. In another embodiment, the region of the target gene having high variability or mutational frequency is identified by obtaining or providing genotype information about the target gene from a population. In another embodiment, the genotype information can be from a population suffering from a liver disorder, such as hepatocellular carcinoma or hepatoblastoma.

Another aspect of the invention features a method of modulating expression, e.g., downregulating or silencing, a target gene, by providing an iRNA agent that has a first sequence and a second sequence sufficiently complementary to each other to hybridize. In addition, the first sequence is complementary to a first target RNA region and the second sequence is complementary to a second target RNA region.

In one embodiment, the iRNA agent is administered to a subject, e.g., a human.

In another embodiment, the first and second sequences are between 15 and 30 nucleotides in length.

In one embodiment, the method of modulating expression of the target gene further includes providing a second iRNA agent that has a third sequence complementary to a third target RNA region. The third sequence can be sufficiently complementary to the first or second sequence to be capable of hybridizing to either the first or second sequence.

Another aspect of the invention features a method of modulating expression, e.g., downregulating or silencing, a plurality of target RNAs, each of the plurality of target RNAs corresponding to a different target gene. The method includes providing an iRNA agent selected by identifying a first region in a first target RNA of the plurality and a second region in a second target RNA of the plurality, where the first and second regions are sufficiently complementary to each other to be capable of hybridizing.

In another aspect of the invention, an iRNA agent molecule includes a first sequence complementary to a first variant RNA target region and a second sequence complementary to a second variant RNA target region, and the first and second variant RNA target regions correspond to first and second variants or mutants of a target gene. In certain embodiments, the target gene is an apoB-100, beta catenin, or glucose-6 phosphatase gene.

In one embodiment, the target gene is a viral gene (e.g., an HCV gene), tumor suppressor or oncogene.

In another embodiment, the first and second variant target RNA regions include allelic variants of the target gene.

In another embodiment, the first and second variant RNA target regions comprise mutations (e.g., point mutations) or polymorphisms of the target gene.

In one embodiment, the first and second variant RNA target regions correspond to hot-spots for genetic variation.

Another aspect of the invention features a plurality (e.g., a panel or bank) of iRNA agents. Each of the iRNA agents of the plurality includes a first sequence complementary to a first variant target RNA region and a second sequence complementary to a second variant target RNA region, where the first and second variant target RNA regions correspond to first and second variants of a target gene. In certain embodiments, the variants are allelic variants of the target gene.

Another aspect of the invention provides a method of identifying an iRNA agent for treating a subject. The method includes providing or obtaining information, e.g., a genotype, about a target gene, providing or obtaining information about a plurality (e.g., panel or bank) of iRNA agents, comparing the information about the target gene to information about the plurality of iRNA agents, and selecting one or more of the plurality of iRNA agents for treating the subject. Each of the plurality of iRNA agents includes a first sequence complementary to a first variant target RNA region and a second sequence complementary to a second variant target RNA region, and the first and second variant target RNA regions correspond to first and second variants of the target gene. The target gene can be an endogenous gene of the subject or a viral gene. The information about the plurality of iRNA agents can be the sequence of the first or second sequence of one or more of the plurality.

In certain embodiments, at least one of the selected iRNA agents includes a sequence capable of hybridizing to an RNA region corresponding to the target gene, and at least one of the selected iRNA agents comprises a sequence capable of hybridizing to an RNA region corresponding to a variant or mutant of the target gene.

In one aspect, the invention relates to compositions and methods for silencing genes expressed in the liver, e.g., to treat disorders of or related to the liver. An iRNA agent composition of the invention can be one which has been modified to alter distribution in favor of the liver.

In another aspect, the invention relates to iRNA agents that can target more than one RNA region, and methods of using and making the iRNA agents. In one embodiment, the RNA is from a gene that is active in the liver, e.g., apoB-100, glucose-6-phosphatase, beta-catenin, or Hepatitis C virus (HCV).

In another aspect, an iRNA agent includes a first and second sequence that are sufficiently complementary to each other to hybridize. The first sequence can be complementary to a first target RNA region and the second sequence can be complementary to a second target RNA region. For example, the first sequence can be complementary to a first target apoB-100 RNA region and the second sequence can be complementary to a second target apoB-100 RNA region.

In one embodiment, the first target RNA region is encoded by a first gene, e.g., a gene expressed in the liver, and the second target RNA region is encoded by a second gene, e.g., a second gene expressed in the liver. In another embodiment, the first and second target RNA regions are different regions of an RNA from a single gene, e.g., a single gene that is at least expressed in the liver. In another embodiment, the first and second sequences differ by at least one and no more than six nucleotides.

In another embodiment, sequence analysis, e.g., to identify common mutants in the target gene, is used to identify a region of the target gene that has high variability or mutational frequency. For example, sequence analysis can be used to identify regions of aopB-100 or beta catenin that have high variability or mutational frequency. In another embodiment, the region of the target gene having high variability or mutational frequency is identified by obtaining or providing genotype information about the target gene from a population. In particular, the genotype information can be from a population suffering from a liver disorder, such as hepatocellular carcinoma or hepatoblastoma.

In another aspect, the invention features a method for reducing apoB-100 levels in a subject, e.g., a mammal, such as a human. The method includes administering to a subject an iRNA agent which targets apoB-100. The iRNA agent can be one described here, and can be a dsRNA that has a sequence that is substantially identical to a sequence of the apoB-100 gene. The iRNA can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.

In a preferred embodiment, the subject is treated with an iRNA agent which targets one of the sequences listed in Tables 5 and 6. In a preferred embodiment it targets both sequences of a palindromic pair provided in Tables 5 and 6. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Tables 5 and 6.

In a preferred embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Tables 5 and 6. In a preferred embodiment the iRNA agent will include regions complementary to the palindromic pairs of Tables 5 and 6 as a duplex region.

In a preferred embodiment the duplex region of the iRNA agent will target a sequence listed in Tables 5 and 6 but will not be perfectly complementary with the target sequence, e.g., it will not be complementary at least 1 base pair. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, in total, or per strand, which do not hybridize with the target sequence

In a preferred embodiment the iRNA agent includes overhangs, e.g., 3′ or 5′ overhangs, preferably one or more 3′ overhangs. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

The iRNA agent that targets apoB-100 can be administered in an amount sufficient to reduce expression of apoB-100 mRNA. In one embodiment, the iRNA agent is administered in an amount sufficient to reduce expression of apoB-100 protein (e.g., by at least 2%, 4%, 6%, 10%, 15%, 20%). Preferably, the iRNA agent does not reduce expression of apoB-48 mRNA or protein. This can be effected, e.g., by selection of an iRNA agent which specifically targets the nucleotides subject to RNA editing in the apoB-100 transcript.

The iRNA agent that targets apoB-100 can be administered to a subject, wherein the subject is suffering from a disorder characterized by elevated or otherwise unwanted expression of apoB-100, elevated or otherwise unwanted levels of cholesterol, and/or disregulation of lipid metabolism. The iRNA agent can be administered to an individual at risk for the disorder to delay onset of the disorder or a symptom of the disorder. These disorders include HDL/LDL cholesterol imbalance; dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), acquired hyperlipidemia; hypercholestorolemia; statin-resistant hypercholesterolemia; coronary artery disease (CAD) coronary heart disease (CHD) atherosclerosis. In one embodiment, the iRNA that targets apoB-100 is administered to a subject suffering from statin-resistant hypercholesterolemia.

The apoB-100 iRNA agent can be administered in an amount sufficient to reduce levels of serum LDL-C and/or HDL-C and/or total cholesterol in a subject. For example, the iRNA is administered in an amount sufficient to decrease total cholesterol by at least 0.5%, 1%, 2.5%, 5%, 10% in the subject. In one embodiment, the iRNA agent is administered in an amount sufficient to reduce the risk of myocardial infarction the subject.

In a preferred embodiment the iRNA agent is administered repeatedly. Administration of an iRNA agent can be carried out over a range of time periods. It can be administered daily, once every few days, weekly, or monthly. The timing of administration can vary from patient to patient, depending on such factors as the severity of a patient's symptoms. For example, an effective dose of an iRNA agent can be administered to a patient once a month for an indefinite period of time, or until the patient no longer requires therapy. In addition, sustained release compositions containing an iRNA agent can be used to maintain a relatively constant dosage in the patient's blood.

In one embodiment, the iRNA agent can be targeted to the liver, and apoB expression level are decreased in the liver following administration of the apoB iRNA agent. For example, the iRNA agent can be complexed with a moiety that targets the liver, e.g., an antibody or ligand that binds a receptor on the liver.

The iRNA agent, particularly an iRNA agent that targets apoB, beta-catenin or glucose-6-phosphatase RNA, can be targeted to the liver, for example by associating, e.g., conjugating the iRNA agent to a lipophilic moiety, e.g., a lipid, cholesterol, oleyl, retinyl, or cholesteryl residue (see Table 1). Other lipophilic moieties that can be associated, e.g., conjugated with the iRNA agent include cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In one embodiment, the iRNA agent can be targeted to the liver by associating, e.g., conjugating, the iRNA agent to a low-density lipoprotein (LDL), e.g., a lactosylated LDL. In another embodiment, the iRNA agent can be targeted to the liver by associating, e.g., conjugating, the iRNA agent to a polymeric carrier complex with sugar residues.

In another embodiment, the iRNA agent can be targeted to the liver by associating, e.g., conjugating, the iRNA agent to a liposome complexed with sugar residues. A targeting agent that incorporates a sugar, e.g., galactose and/or analogues thereof, is particularly useful. These agents target, in particular, the parenchymal cells of the liver (see Table 1). In a preferred embodiment, the targeting moiety includes more than one galactose moiety, preferably two or three. Preferably, the targeting moiety includes 3 galactose moieties, e.g., spaced about 15 angstroms from each other. The targeting moiety can be lactose. A lactose is a glucose coupled to a galactose. Preferably, the targeting moiety includes three lactoses. The targeting moiety can also be N-Acetyl-Galactosamine, N-Ac-Glucosamine. A mannose, or mannose-6-phosphate targeting moiety can be used for macrophage targeting.

The targeting agent can be linked directly, e.g., covalently or non covalently, to the iRNA agent, or to another delivery or formulation modality, e.g., a liposome. E.g., the iRNA agents with or without a targeting moiety can be incorporated into a delivery modality, e.g., a liposome, with or without a targeting moiety.

It is particularly preferred to use an iRNA conjugated to a lipophilic molecule to conjugate to an iRNA agent that targets apoB, beta-catenin or glucose-6-phosphatase iRNA targeting agent.

In one embodiment, the iRNA agent has been modified, or is associated with a delivery agent, e.g., a delivery agent described herein, e.g., a liposome, which has been modified to alter distribution in favor of the liver. In one embodiment, the modification mediates association with a serum albumin (SA), e.g., a human serum albumin (HSA), or a fragment thereof.

The iRNA agent, particularly an iRNA agent that targets apoB, beta-catenin or glucose-6-phosphatase RNA, can be targeted to the liver, for example by associating, e.g., conjugating the iRNA agent to an SA molecule, e.g., an HSA molecule, or a fragment thereof. In one embodiment, the iRNA agent or composition thereof has an affinity for an SA, e.g., HSA, which is sufficiently high such that its levels in the liver are at least 10, 20, 30, 50, or 100% greater in the presence of SA, e.g., HSA, or is such that addition of exogenous SA will increase delivery to the liver. These criteria can be measured, e.g., by testing distribution in a mouse in the presence or absence of exogenous mouse or human SA.

The SA, e.g., HSA, targeting agent can be linked directly, e.g., covalently or non-covalently, to the iRNA agent, or to another delivery or formulation modality, e.g., a liposome. E.g., the iRNA agents with or without a targeting moiety can be incorporated into a delivery modality, e.g., a liposome, with or without a targeting moiety.

It is particularly preferred to use an iRNA conjugated to an SA, e.g., an HSA, molecule wherein the iRNA agent is an apoB, beta-catenin or glucose-6-phosphatase iRNA targeting agent.

In another aspect, the invention features, a method for reducing glucose-6-phosphatase levels in a subject, e.g., a mammal, such as a human. The method includes administering to a subject an iRNA agent which targets glucose-6-phosphatase. The iRNA agent can be a dsRNA that has a sequence that is substantially identical to a sequence of the glucose-6-phosphatase gene.

In a preferred embodiment, the subject is treated with an iRNA agent which targets one of the sequences listed in Table 7. In a preferred embodiment it targets both sequences of a palindromic pair provided in Table 7. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Table 7.

In a preferred embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Table 7. In a preferred embodiment the iRNA agent will include regions complementary to the palindromic pairs of Table 7 as a duplex region.

In a preferred embodiment the duplex region of the iRNA agent will target a sequence listed in Table 7_but will not be perfectly complementary with the target sequence, e.g., it will not be complementary at least 1 base pair. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, in total, or per strand, which do not hybridize with the target sequence

In a preferred embodiment the iRNA agent includes overhangs, e.g., 3′ or 5′ overhangs, preferably one or more 3′ overhangs. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

Table 7 refers to sequences from human glucose-6-phosphatase. Table 8 refers to sequences from rat glucose-6-phosphatase. The sequences from table 8 can be used, e.g., in experiments with rats or cultured rat cells.

In a preferred embodiment iRNA agent can have any architecture, e.g., architecture described herein. E.g., it can be incorporated into an iRNA agent having an overhang structure, overall length, hairpin vs. two-strand structure, as described herein. In addition, monomers other than naturally occurring ribonucleotides can be used in the selected iRNA agent.

The iRNA that targets glucose-6-phosphatase can be administered in an amount sufficient to reduce expression of glucose-6-phosphatase mRNA.

The iRNA that targets glucose-6-phosphatase can be administered to a subject to inhibit hepatic glucose production, for the treatment of glucose-metabolism-related disorders, such as diabetes, e.g., type-2-diabetes mellitus. The iRNA agent can be administered to an individual at risk for the disorder to delay onset of the disorder or a symptom of the disorder.

In other embodiments, iRNA agents having sequence similarity to the following genes can also be used to inhibit hepatic glucose production. These other genes include “forkhead homologue in rhabdomyosarcoma (FKHR); glucagon; glucagon receptor; glycogen phosphorylase; PPAR-Gamma Coactivator (PGC-1); Fructose-1,6-bisphosphatase; glucose-6-phosphate locator; glucokinase inhibitory regulatory protein; and phosphoenolpyruvate carboxykinase (PEPCK).

In one embodiment, the iRNA agent can be targeted to the liver, and RNA expression levels of the targeted genes are decreased in the liver following administration of the iRNA agent.

The iRNA agent can be one described herein, and can be a dsRNA that has a sequence that is substantially identical to a sequence of a target gene. The iRNA can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length

In another aspect, the invention features a method for reducing beta-catenin levels in a subject, e.g., a mammal, such as a human. The method includes administering to a subject an iRNA agent that targets beta-catenin. The iRNA agent can be one described herein, and can be a dsRNA that has a sequence that is substantially identical to a sequence of the beta-catenin gene. The iRNA can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.

In a preferred embodiment, the subject is treated with an iRNA agent which targets one of the sequences listed in Table 9. In a preferred embodiment it targets both sequences of a palindromic pair provided in Table 9. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Table 9.

In a preferred embodiment, the subject is treated with an iRNA agent which targets one of the sequences listed in Table 9. In a preferred embodiment it targets both sequences of a palindromic pair provided in Table 9. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Table 9.

In a preferred embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Table 9. In a preferred embodiment the iRNA agent will include regions complementary to the palindromic pairs of Table 9 as a duplex region.

In a preferred embodiment the duplex region of the iRNA agent will target a sequence listed in Table 9 but will not be perfectly complementary with the target sequence, e.g., it will not be complementary at least 1 base pair. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, in total, or per strand, which do not hybridize with the target sequence

In a preferred embodiment the iRNA agent includes overhangs, e.g., 3′ or 5′ overhangs, preferably one or more 3′ overhangs. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

The iRNA agent that targets beta-catenin can be administered in an amount sufficient to reduce expression of beta-catenin mRNA. In one embodiment, the iRNA agent is administered in an amount sufficient to reduce expression of beta-catenin protein (e.g., by at least 2%, 4%, 6%, 10%, 15%, 20%).

The iRNA agent that targets beta-catenin can be administered to a subject, wherein the subject is suffering from a disorder characterized by unwanted cellular proliferation in the liver or of liver tissue, e.g., metastatic tissue originating from the liver. Examples include, a benign or malignant disorder, e.g., a cancer, e.g., a hepatocellular carcinoma (HCC), hepatic metastasis, or hepatoblastoma.

The iRNA agent can be administered to an individual at risk for the disorder to delay onset of the disorder or a symptom of the disorder

In a preferred embodiment the iRNA agent is administered repeatedly. Administration of an iRNA agent can be carried out over a range of time periods. It can be administered daily, once every few days, weekly, or monthly. The timing of administration can vary from patient to patient, depending on such factors as the severity of a patient's symptoms. For example, an effective dose of an iRNA agent can be administered to a patient once a month for an indefinite period of time, or until the patient no longer requires therapy. In addition, sustained release compositions containing an iRNA agent can be used to maintain a relatively constant dosage in the patient's blood.

In one embodiment, the iRNA agent can be targeted to the liver, and beta-catenin expression level are decreased in the liver following administration of the beta-catenin iRNA agent. For example, the iRNA agent can be complexed with a moiety that targets the liver, e.g., an antibody or ligand that binds a receptor on the liver.

In another aspect, the invention provides methods to treat liver disorders, e.g., disorders characterized by unwanted cell proliferation, hematological disorders, disorders characterized by inflammation disorders, and metabolic or viral diseases or disorders of the liver. A proliferation disorder of the liver can be, for example, a benign or malignant disorder, e.g., a cancer, e.g, a hepatocellular carcinoma (HCC), hepatic metastasis, or hepatoblastoma. A hepatic hematology or inflammation disorder can be a disorder involving clotting factors, a complement-mediated inflammation or a fibrosis, for example. Metabolic diseases of the liver can include dyslipidemias, and irregularities in glucose regulation. Viral diseases of the liver can include hepatitis C or hepatitis B. In one embodiment, a liver disorder is treated by administering one or more iRNA agents that have a sequence that is substantially identical to a sequence in a gene involved in the liver disorder.

In one embodiment an iRNA agent to treat a liver disorder has a sequence which is substantially identical to a sequence of the beta-catenin or c-jun gene. In another embodiment, such as for the treatment of hepatitis C or hepatitis B, the iRNA agent can have a sequence that is substantially identical to a sequence of a gene of the hepatitis C virus or the hepatitis B virus, respectively. For example, the iRNA agent can target the 5′ core region of HCV. This region lies just downstream of the ribosomal toe-print straddling the initiator methionine. Alternatively, an iRNA agent of the invention can target any one of the nonstructural proteins of HCV: NS3, 4A, 4B, 5A, or 5B. For the treatment of hepatitis B, an iRNA agent can target the protein X (HBx) gene, for example.

In a preferred embodiment, the subject is treated with an iRNA agent which targets one of the sequences listed in Table 10. In a preferred embodiment it targets both sequences of a palindromic pair provided in Table 10. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Table 10.

In a preferred embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Table 10. In a preferred embodiment the iRNA agent will include regions complementary to the palindromic pairs of Table 10 as a duplex region.

In a preferred embodiment the duplex region of the iRNA agent will target a sequence listed in Table 10, but will not be perfectly complementary with the target sequence, e.g., it will not be complementary at least 1 base pair. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, in total, or per strand, which do not hybridize with the target sequence

In a preferred embodiment the iRNA agent includes overhangs, e.g., 3′ or 5′ overhangs, preferably one or more 3′ overhangs. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

In another aspect, an iRNA agent can be administered to modulate blood clotting, e.g., to reduce the tendency to form a blood clot. In a preferred embodiment the iRNA agent targets Factor V expression, preferably in the liver. One or more iRNA agents can be used to target a wild type allele, a mutant allele, e.g., the Leiden Factor V allele, or both. Such administration can be used to treat or prevent venous thrombosis, e.g., deep vein thrombosis or pulmonary embolism, or another disorder caused by elevated or otherwise unwanted expression of Factor V, in, e.g., the liver. In one embodiment the iRNA agent can treat a subject, e.g., a human who has Factor V Leiden or other genetic trait associated with an unwanted tendency to form blood clots.

In a preferred embodiment administration of an iRNA agent which targets Factor V is with the administration of a second treatment, e.g, a treatment which reduces the tendency of the blood to clot, e.g., the administration of heparin or of a low molecular weight heparin.

In one embodiment, the iRNA agent that targets Factor V can be used as a prophylaxis in patients, e.g., patients with Factor V Leiden, who are placed at risk for a thrombosis, e.g., those about to undergo surgery, in particular those about to undergo high-risk surgical procedures known to be associated with formation of venous thrombosis, those about to undergo a prolonged period of relative inactivity, e.g., on a motor vehicle, train or airplane flight, e.g., a flight or other trip lasting more than three or five hours. Such a treatment can be an adjunct to the therapeutic use of low molecular weight (LMW) heparin prophylaxis.

In another embodiment, the iRNA agent that targets Factor V can be administered to patients with Factor V Leiden to treat deep vein thrombosis (DVT) or pulmonary embolism (PE). Such a treatment can be an adjunct to (or can replace) therapeutic uses of heparin or coumadin. The treatment can be administered by inhalation or generally by pulmonary routes.

In a preferred embodiment, an iRNA agent administered to treat a liver disorder is targeted to the liver. For example, the iRNA agent can be complexed with a targeting moiety, e.g., an antibody or ligand that recognizes a liver-specific receptor.

The invention also includes preparations, including substantially pure or pharmaceutically acceptable preparations of iRNA agents which silence any of the genes discussed herein and in particular for any of apoB-100, glucose-6-phosphatase, beta-catenin, factor V, or any of the HVC genes discussed herein.

The methods and compositions of the invention, e.g., the methods and compositions to treat diseases and disorders of the liver described herein, can be used with any of the iRNA agents described. In addition, the methods and compositions of the invention can be used for the treatment of any disease or disorder described herein, and for the treatment of any subject, e.g., any animal, any mammal, such as any human.

In another aspect, the invention features, a method of selecting two sequences or strands for use in an iRNA agent. The method includes:

-   -   providing a first candidate sequence and a second candidate         sequence;     -   determining the value of a parameter which is a function of the         number of palindromic pairs between the first and second         sequence, wherein a palindromic pair is a nucleotide on said         first sequence which, when the sequences are aligned in         anti-parallel orientation, will hybridize with a nucleotide on         said second sequence;     -   comparing the number with a predetermined reference value, and         if the number has a predetermined relationship with the         reference, e.g., if it is the same or greater, selecting the         sequences for use in an iRNA agent. In most cases each of the         two sequences will be completely complementary with a target         sequence (though as described elsewhere herein that may not         always be the case, there may not be perfect complementarity         with one or both of the target sequences) and will have         sufficient complementarity with each other to form a duplex. The         parameter can be derived e.g., by directly determining the         number of palindromic pairs, e.g., by inspection or by the use         of a computer program which compares or analyses sequence. The         parameter can also be determined less directly, and include         e.g., calculation of or measurement of the Tm or other value         related to the free energy of association or dissociation of a         duplex.

In a preferred embodiment the determination can be performed on a target sequence, e.g., a genomic sequence. In such embodiments the selected sequence is converted to its complement in the iRNA agent.

In a preferred embodiment the first and second sequences are selected from the sequence of a single target gene. In other embodiments the first sequence is selected from the sequence of a first target gene and the second sequence is selected from the target of a second target gene.

In a preferred embodiment the method includes comparing blocks of sequence, e.g., blocks which are between 15 and 25 nucleotides in length, and preferably 19, 20, or 21, and most preferably 19 nucleotides in length, to determine if they are suitable for use, e.g., if they possess sufficient palindromic pairs.

In a preferred embodiment the first and second sequences are divided into a plurality of regions, e.g., terminal regions and a middle region disposed between the terminal regions and where in the reference value, or the predetermined relationship to the reference value, is different for at least two regions. E.g., the first and second sequences, when aligned in anti-parallel orientation, are divided into terminal regions each of a selected number of base pairs, e.g., 2, 3, 4, 5, or 6, and a middle region, and the reference value for the terminal regions is higher than for the middle regions. In other words, a higher number or proportion of palindromic pairs is required in the terminal regions.

In a preferred embodiment the first and second sequences are gene sequences thus the complements of the sequences will be used in a iRNA agent.

In a preferred embodiment hybridize means a classical Watson-Crick pairing. In other embodiments hybridize can include non-Watson-Crick paring, e.g., parings seen in micro RNA precursors.

In a preferred embodiment the method includes the addition of nucleotides to form overhangs, e.g., 3′ or 5′ overhangs, preferably one or more 3′ overhangs. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

In a preferred embodiment the method is used to select all or part of a iRNA agent. The selected sequences can be incorporated into an iRNA agent having any architecture, e.g., an architecture described herein. E.g., it can be incorporated into an iRNA agent having an overhang structure, overall length, hairpin vs. two-strand structure, as described herein. In addition, monomers other than naturally occurring ribonucleotides can be used in the selected iRNA agent.

Preferred iRNA agents of this method will target genes expressed in the liver, e.g., one of the genes disclosed herein, e.g., apo B, Beta catenin, an HVC gene, or glucose 6 phosphatase.

In another aspect, the invention features, an iRNA agent, determined, made, or selected by a method described herein.

The methods and compositions of the invention, e.g., the methods and iRNA compositions to treat liver-based diseases described herein, can be used with any dosage and/or formulation described herein, as well as with any route of administration described herein.

The invention also provides for the use of an iRNA agent which includes monomers which can form other than a canonical Watson-Crick pairing with another monomer, e.g., a monomer on another strand.

The use of “other than canonical Watson-Crick pairing” between monomers of a duplex can be used to control, often to promote, melting of all or part of a duplex. The iRNA agent can include a monomer at a selected or constrained position that results in a first level of stability in the iRNA agent duplex (e.g., between the two separate molecules of a double stranded iRNA agent) and a second level of stability in a duplex between a sequence of an iRNA agent and another sequence molecule, e.g., a target or off-target sequence in a subject. In some cases the second duplex has a relatively greater level of stability, e.g., in a duplex between an anti-sense sequence of an iRNA agent and a target mRNA. In this case one or more of the monomers, the position of the monomers in the iRNA agent, and the target sequence (sometimes referred to herein as the selection or constraint parameters), are selected such that the iRNA agent duplex is has a comparatively lower free energy of association (which while not wishing to be bound by mechanism or theory, is believed to contribute to efficacy by promoting disassociation of the duplex iRNA agent in the context of the RISC) while the duplex formed between an anti-sense targeting sequence and its target sequence, has a relatively higher free energy of association (which while not wishing to be bound by mechanism or theory, is believed to contribute to efficacy by promoting association of the anti-sense sequence and the target RNA).

In other cases the second duplex has a relatively lower level of stability, e.g., in a duplex between a sense sequence of an iRNA agent and an off-target mRNA. In this case one or more of the monomers, the position of the monomers in the iRNA agent, and an off-target sequence, are selected such that the iRNA agent duplex is has a comparatively higher free energy of association while the duplex formed between a sense targeting sequence and its off-target sequence, has a relatively lower free energy of association (which while not wishing to be bound by mechanism or theory, is believed to reduce the level of off-target silencing by contribute to efficacy by promoting disassociation of the duplex formed by the sense strand and the off-target sequence).

Thus, inherent in the structure of the iRNA agent is the property of having a first stability for the intra-iRNA agent duplex and a second stability for a duplex formed between a sequence from the iRNA agent and another RNA, e.g., a target mRNA. As discussed above, this can be accomplished by judicious selection of one or more of the monomers at a selected or constrained position, the selection of the position in the duplex to place the selected or constrained position, and selection of the sequence of a target sequence (e.g., the particular region of a target gene which is to be targeted). The iRNA agent sequences which satisfy these requirements are sometimes referred herein as constrained sequences. Exercise of the constraint or selection parameters can be, e.g., by inspection, or by computer assisted methods. Exercise of the parameters can result in selection of a target sequence and of particular monomers to give a desired result in terms of the stability, or relative stability, of a duplex.

Thus, in one aspect, the invention features, an iRNA agent which includes: a first sequence which targets a first target region and a second sequence which targets a second target region. The first and second sequences have sufficient complementarity to each other to hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In a duplex region of the iRNA agent, at a selected or constrained position, the first target region has a first monomer, and the second target region has a second monomer. The first and second monomers occupy complementary or corresponding positions. One, and preferably both monomers are selected such that the stability of the pairing of the monomers contribute to a duplex between the first and second sequence will differ form the stability of the pairing between the first or second sequence with a target sequence.

Usually, the monomers will be selected (selection of the target sequence may be required as well) such that they form a pairing in the iRNA agent duplex which has a lower free energy of dissociation, and a lower Tm, than will be possessed by the paring of the monomer with its complementary monomer in a duplex between the iRNA agent sequence and a target RNA duplex.

The constraint placed upon the monomers can be applied at a selected site or at more than one selected site. By way of example, the constraint can be applied at more than 1, but less than 3, 4, 5, 6, or 7 sites in an iRNA agent duplex.

A constrained or selected site can be present at a number of positions in the iRNA agent duplex. E.g., a constrained or selected site can be present within 3, 4, 5, or 6 positions from either end, 3′ or 5′ of a duplexed sequence. A constrained or selected site can be present in the middle of the duplex region, e.g., it can be more than 3, 4, 5, or 6, positions from the end of a duplexed region.

The iRNA agent can be selected to target a broad spectrum of genes, including any of the genes described herein.

In a preferred embodiment the iRNA agent has an architecture (architecture refers to one or more of overall length, length of a duplex region, the presence, number, location, or length of overhangs, sing strand versus double strand form) described herein.

E.g., the iRNA agent can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length and there is a duplex region of about 19 pairs. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.

In some embodiment the duplex region of the iRNA agent will have, mismatches, in addition to the selected or constrained site or sites. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, which do not form canonical Watson-Crick pairs or which do not hybridize. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

The monomers can be selected such that: first and second monomers are naturally occurring ribonucleotides, or modified ribonucleotides having naturally occurring bases, and when occupying complementary sites either do not pair and have no substantial level of H-bonding, or form a non canonical Watson-Crick pairing and form a non-canonical pattern of H bonding, which usually have a lower free energy of dissociation than seen in a canonical Watson-Crick pairing, or otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing. When one (or both) of the iRNA agent sequences duplexes with a target, the first (or second) monomer forms a canonical Watson-Crick pairing with the base in the complementary position on the target, or forms a non canonical Watson-Crick pairing having a higher free energy of dissociation and a higher Tm than seen in the paring in the iRNA agent. The classical Watson-Crick parings are as follows: A-T, G-C, and A-U. Non-canonical Watson-Crick pairings are known in the art and can include, U-U, G-G, G-Atrans, G-Acis, and GU.

The monomer in one or both of the sequences is selected such that, it does not pair, or forms a pair with its corresponding monomer in the other sequence which minimizes stability (e.g., the H bonding formed between the monomer at the selected site in the one sequence and its monomer at the corresponding site in the other sequence are less stable than the H bonds formed by the monomer one (or both) of the sequences with the respective target sequence. The monomer in one or both strands is also chosen to promote stability in one or both of the duplexes made by a strand and its target sequence. E.g., one or more of the monomers and the target sequences are selected such that at the selected or constrained position, there is are no H bonds formed, or a non canonical pairing is formed in the iRNA agent duplex, or otherwise they otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing, but when one (or both) sequences form a duplex with the respective target, the pairing at the selected or constrained site is a canonical Watson-Crick pairing.

The inclusion of such a monomers will have one or more of the following effects: it will destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes referred to as off-target sequences, and duplex interactions between the a sequence and the intended target will not be destabilized.

By way of example:

the monomer at the selected site in the first sequence includes an A (or a modified base which pairs with T), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., G. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.

the monomer at the selected site in the first sequence includes U (or a modified base which pairs with A), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., U or G. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.

The monomer at the selected site in the first sequence includes a G (or a modified base which pairs with C), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., G, Acis, Atrans, or U. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.

The monomer at the selected site in the first sequence includes a C (or a modified base which pairs with G), and the monomer in at the selected position in the second sequence is chosen a monomer which will not pair or which will form a non-canonical pairing. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.

In another embodiment a non-naturally occurring or modified monomer or monomers are chosen such that when a non-naturally occurring or modified monomer occupies a positions at the selected or constrained position in an iRNA agent they exhibit a first free energy of dissociation and when one (or both) of them pairs with a naturally occurring monomer, the pair exhibits a second free energy of dissociation, which is usually higher than that of the pairing of the first and second monomers. E.g., when the first and second monomers occupy complementary positions they either do not pair and have no substantial level of H-bonding, or form a weaker bond than one of them would form with a naturally occurring monomer, and reduce the stability of that duplex, but when the duplex dissociates at least one of the strands will form a duplex with a target in which the selected monomer will promote stability, e.g., the monomer will form a more stable pair with a naturally occurring monomer in the target sequence than the pairing it formed in the iRNA agent.

An example of such a pairing is 2-amino A and either of a 2-thio pyrimidine analog of U or T.

When placed in complementary positions of the iRNA agent these monomers will pair very poorly and will minimize stability. However, a duplex is formed between 2 amino A and the U of a naturally occurring target, or a duplex is between 2-thio U and the A of a naturally occurring target or 2-thio T and the A of a naturally occurring target will have a relatively higher free energy of dissociation and be more stable. This is shown in the FIG. 1.

The pair shown in FIG. 1 (the 2-amino A and the 2-s U and T) is exemplary. In another embodiment, the monomer at the selected position in the sense strand can be a universal pairing moiety. A universal pairing agent will form some level of H bonding with more than one and preferably all other naturally occurring monomers. An example of a universal pairing moiety is a monomer which includes 3-nitro pyrrole. (Examples of other candidate universal base analogs can be found in the art, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incorporated by reference. Examples can also be found in the section on Universal Bases below.) In these cases the monomer at the corresponding position of the anti-sense strand can be chosen for its ability to form a duplex with the target and can include, e.g., A, U, G, or C.

In another aspect, the invention features, an iRNA agent which includes: a sense sequence, which preferably does not target a sequence in a subject, and an anti-sense sequence, which targets a target gene in a subject. The sense and anti-sense sequences have sufficient complementarity to each other to hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In a duplex region of the iRNA agent, at a selected or constrained position, the monomers are selected such that:

the monomer in the sense sequence is selected such that, it does not pair, or forms a pair with its corresponding monomer in the anti-sense strand which minimizes stability (e.g., the H bonding formed between the monomer at the selected site in the sense strand and its monomer at the corresponding site in the anti-sense strand are less stable than the H bonds formed by the monomer of the anti-sense sequence and its canonical Watson-Crick partner or, if the monomer in the anti-sense strand includes a modified base, the natural analog of the modified base and its canonical Watson-Crick partner);

the monomer is in the corresponding position in the anti-sense strand is selected such that it maximizes the stability of a duplex it forms with the target sequence, e.g., it forms a canonical Watson-Crick paring with the monomer in the corresponding position on the target stand;

optionally, the monomer in the sense sequence is selected such that, it does not pair, or forms a pair with its corresponding monomer in the anti-sense strand which minimizes stability with an off-target sequence.

The inclusion of such a monomers will have one or more of the following effects: it will destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes referred to as off-target sequences, and duplex interactions between the anti-sense strand and the intended target will not be destabilized.

The constraint placed upon the monomers can be applied at a selected site or at more than one selected site. By way of example, the constraint can be applied at more than 1, but less than 3, 4, 5, 6, or 7 sites in an iRNA agent duplex.

A constrained or selected site can be present at a number of positions in the iRNA agent duplex. E.g., a constrained or selected site can be present within 3, 4, 5, or 6 positions from either end, 3′ or 5′ of a duplexed sequence. A constrained or selected site can be present in the middle of the duplex region, e.g., it can be more than 3, 4, 5, or 6, positions from the end of a duplexed region.

The iRNA agent can be selected to target a broad spectrum of genes, including any of the genes described herein.

In a preferred embodiment the iRNA agent has an architecture (architecture refers to one or more of overall length, length of a duplex region, the presence, number, location, or length of overhangs, sing strand versus double strand form) described herein.

E.g., the iRNA agent can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length and there is a duplex region of about 19 pairs. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.

In some embodiment the duplex region of the iRNA agent will have, mismatches, in addition to the selected or constrained site or sites. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, which do not form canonical Watson-Crick pairs or which do not hybridize. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

One or more selection or constraint parameters can be exercised such that: monomers at the selected site in the sense and anti-sense sequences are both naturally occurring ribonucleotides, or modified ribonucleotides having naturally occurring bases, and when occupying complementary sites in the iRNA agent duplex either do not pair and have no substantial level of H-bonding, or form a non-canonical Watson-Crick pairing and thus form a non-canonical pattern of H bonding, which generally have a lower free energy of dissociation than seen in a Watson-Crick pairing, or otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing. When one, usually the anti-sense sequence of the iRNA agent sequences forms a duplex with another sequence, generally a sequence in the subject, and generally a target sequence, the monomer forms a classic Watson-Crick pairing with the base in the complementary position on the target, or forms a non-canonical Watson-Crick pairing having a higher free energy of dissociation and a higher Tm than seen in the paring in the iRNA agent. Optionally, when the other sequence of the iRNA agent, usually the sense sequences forms a duplex with another sequence, generally a sequence in the subject, and generally an off-target sequence, the monomer fails to forms a canonical Watson-Crick pairing with the base in the complementary position on the off target sequence, e.g., it forms or forms a non-canonical Watson-Crick pairing having a lower free energy of dissociation and a lower Tm.

By way of example:

the monomer at the selected site in the anti-sense stand includes an A (or a modified base which pairs with T), the corresponding monomer in the target is a T, and the sense strand is chosen from a base which will not pair or which will form a noncanonical pair, e.g., G;

the monomer at the selected site in the anti-sense stand includes a U (or a modified base which pairs with A), the corresponding monomer in the target is an A, and the sense strand is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., U or G;

the monomer at the selected site in the anti-sense stand includes a C (or a modified base which pairs with G), the corresponding monomer in the target is a G, and the sense strand is chosen a monomer which will not pair or which will form a non-canonical pairing, e.g., G, A_(cis), A_(trans), or U; or

the monomer at the selected site in the anti-sense stand includes a G (or a modified base which pairs with C), the corresponding monomer in the target is a C, and the sense strand is chosen from a monomer which will not pair or which will form a non-canonical pairing.

In another embodiment a non-naturally occurring or modified monomer or monomers is chosen such that when it occupies complementary a position in an iRNA agent they exhibit a first free energy of dissociation and when one (or both) of them pairs with a naturally occurring monomer, the pair exhibits a second free energy of dissociation, which is usually higher than that of the pairing of the first and second monomers. E.g., when the first and second monomers occupy complementary positions they either do not pair and have no substantial level of H-bonding, or form a weaker bond than one of them would form with a naturally occurring monomer, and reduce the stability of that duplex, but when the duplex dissociates at least one of the strands will form a duplex with a target in which the selected monomer will promote stability, e.g., the monomer will form a more stable pair with a naturally occurring monomer in the target sequence than the pairing it formed in the iRNA agent.

An example of such a pairing is 2-amino A and either of a 2-thio pyrimidine analog of U or T. As is discussed above, when placed in complementary positions of the iRNA agent these monomers will pair very poorly and will minimize stability. However, a duplex is formed between 2 amino A and the U of a naturally occurring target, or a duplex is formed between 2-thio U and the A of a naturally occurring target or 2-thio T and the A of a naturally occurring target will have a relatively higher free energy of dissociation and be more stable.

The monomer at the selected position in the sense strand can be a universal pairing moiety. A universal pairing agent will form some level of H bonding with more than one and preferably all other naturally occurring monomers. An examples of a universal pairing moiety is a monomer which includes 3-nitro pyrrole. Examples of other candidate universal base analogs can be found in the art, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incorporated by reference. In these cases the monomer at the corresponding position of the anti-sense strand can be chosen for its ability to form a duplex with the target and can include, e.g., A, U, G, or C.

In another aspect, the invention features, an iRNA agent which includes: a sense sequence, which preferably does not target a sequence in a subject, and an anti-sense sequence, which targets a plurality of target sequences in a subject, wherein the targets differ in sequence at only 1 or a small number, e.g., no more than 5, 4, 3 or 2 positions. The sense and anti-sense sequences have sufficient complementarity to each other to hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In the sequence of the anti-sense strand of the iRNA agent is selected such that at one, some, or all of the positions which correspond to positions that differ in sequence between the target sequences, the anti-sense strand will include a monomer which will form H-bonds with at least two different target sequences. In a preferred example the anti-sense sequence will include a universal or promiscuous monomer, e.g., a monomer which includes 5-nitro pyrrole, 2-amino A, 2-thio U or 2-thio T, or other universal base referred to herein.

In a preferred embodiment the iRNA agent targets repeated sequences (which differ at only one or a small number of positions from each other) in a single gene, a plurality of genes, or a viral genome, e.g., the HCV genome.

An embodiment is illustrated in the FIGS. 2 and 3.

In another aspect, the invention features, determining, e.g., by measurement or calculation, the stability of a pairing between monomers at a selected or constrained position in the iRNA agent duplex, and preferably determining the stability for the corresponding pairing in a duplex between a sequence form the iRNA agent and another RNA, e.g., a target sequence. The determinations can be compared. An iRNA agent thus analyzed can be used in the development of a further modified iRNA agent or can be administered to a subject. This analysis can be performed successively to refine or design optimized iRNA agents.

In another aspect, the invention features, a kit which includes one or more of the following an iRNA described herein, a sterile container in which the iRNA agent is disclosed, and instructions for use.

In another aspect, the invention features, an iRNA agent containing a constrained sequence made by a method described herein. The iRNA agent can target one or more of the genes referred to herein.

iRNA agents having constrained or selected sites, e.g., as described herein, can be used in any way described herein. Accordingly, they iRNA agents having constrained or selected sites, e.g., as described herein, can be used to silence a target, e.g., in any of the methods described herein and to target any of the genes described herein or to treat any of the disorders described herein. iRNA agents having constrained or selected sites, e.g., as described herein, can be incorporated into any of the formulations or preparations, e.g., pharmaceutical or sterile preparations described herein. iRNA agents having constrained or selected sites, e.g., as described herein, can be administered by any of the routes of administration described herein.

The term “other than canonical Watson-Crick pairing” as used herein, refers to a pairing between a first monomer in a first sequence and a second monomer at the corresponding position in a second sequence of a duplex in which one or more of the following is true: (1) there is essentially no pairing between the two, e.g., there is no significant level of H bonding between the monomers or binding between the monomers does not contribute in any significant way to the stability of the duplex; (2) the monomers are a non-canonical paring of monomers having a naturally occurring bases, i.e., they are other than A-T, A-U, or G-C, and they form monomer-monomer H bonds, although generally the H bonding pattern formed is less strong than the bonds formed by a canonical pairing; or (3) at least one of the monomers includes a non-naturally occurring bases and the H bonds formed between the monomers is, preferably formed is less strong than the bonds formed by a canonical pairing, namely one or more of A-T, A-U, G-C.

The term “off-target” as used herein, refers to a sequence other than the sequence to be silenced.

Universal Bases: “wild-cards”; shape-based complementarity

Bi-stranded, multisite replication of a base pair between difluorotoluene and adenine: confirmation by ‘inverse’ sequencing. Liu, D.; Moran, S.; Kool, E. T. Chem. Biol., 1997, 4, 919-926)

(Importance of terminal base pair hydrogen-bonding in 3′-end proofreading by the Klenow fragment of DNA polymerase I. Morales, J. C.; Kool, E. T. Biochemistry, 2000, 39, 2626-2632)

(Selective and stable DNA base pairing without hydrogen bonds. Matray, T, J.; Kool, E. T. J. Am. Chem. Soc., 1998, 120, 6191-6192)

(Difluorotoluene, a nonpolar isostere for thymine, codes specifically and efficiently for adenine in DNA replication. Moran, S. Ren, R. X.-F.; Rumney IV, S.; Kool, E. T. J. Am. Chem. Soc., 1997, 119, 2056-2057)

(Structure and base pairing properties of a replicable nonpolar isostere for deoxyadenosine. Guckian, K. M.; Morales, J. C.; Kool, E. T. J. Org. Chem., 1998, 63, 9652-9656)

(Universal bases for hybridization, replication and chain termination. Berger, M.; Wu. Y.; Ogawa, A. K.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. Nucleic Acids Res., 2000, 28, 2911-2914)

(1. Efforts toward the expansion of the genetic alphabet: Information storage and replication with unnatural hydrophobic base pairs. Ogawa, A. K.; Wu, Y.; McMinn, D. L.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 3274-3287. 2. Rational design of an unnatural base pair with increased kinetic selectivity. Ogawa, A. K.; Wu. Y.; Berger, M.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 8803-8804)

(Efforts toward expansion of the genetic alphabet: replication of DNA with three base pairs. Tae, E. L.; Wu, Y.; Xia, G.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2001, 123, 7439-7440)

(1. Efforts toward expansion of the genetic alphabet: Optimization of interbase hydrophobic interactions. Wu, Y.; Ogawa, A. K.; Berger, M.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 7621-7632. 2. Efforts toward expansion of genetic alphabet: DNA polymerase recognition of a highly stable, self-pairing hydrophobic base. McMinn, D. L.; Ogawa. A. K.; Wu, Y.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 1999, 121, 11585-11586)

(A stable DNA duplex containing a non-hydrogen-bonding and non-shape complementary base couple: Interstrand stacking as the stability determining factor. Brotschi, C.; Haberli, A.; Leumann, C, J. Angew. Chem. Int. Ed., 2001, 40, 3012-3014)

(2,2′-Bipyridine Ligandoside: A novel building block for modifying DNA with intra-duplex metal complexes. Weizman, H.; Tor, Y. J. Am. Chem. Soc., 2001, 123, 3375-3376)

(Minor groove hydration is critical to the stability of DNA duplexes. Lan, T.; McLaughlin, L. W. J. Am. Chem. Soc., 2000, 122, 6512-13)

(Effect of the Universal base 3-nitropyrrole on the selectivity of neighboring natural bases. Oliver, J. S.; Parker, K. A.; Suggs, J. W. Organic Lett., 2001, 3, 1977-1980. 2. Effect of the 1-(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrol residue on the stability of DNA duplexes and triplexes. Amosova, O.; George J.; Fresco, J. R. Nucleic Acids Res., 1997, 25, 1930-1934. 3. Synthesis, structure and deoxyribonucleic acid sequencing with a universal nucleosides: 1-(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrole. Bergstrom, D. E.; Zhang, P.; Toma, P. H.; Andrews, P. C.; Nichols, R. J. Am. Chem. Soc., 1995, 117, 1201-1209)

(Model studies directed toward a general triplex DNA recognition scheme: a novel DNA base that binds a CG base-pair in an organic solvent. Zimmerman, S. C.; Schmitt, P. J. Am. Chem. Soc., 1995, 117, 10769-10770)

(A universal, photocleavable DNA base: nitropiperonyl 2′-deoxyriboside. J. Org. Chem., 2001, 66, 2067-2071)

(Recognition of a single guanine bulge by 2-acylamino-1,8-naphthyridine. Nakatani, K.; Sando, S.; Saito, I. J. Am. Chem. Soc., 2000, 122, 2172-2177. b. Specific binding of 2-amino-1,8-naphthyridine into single guanine bulge as evidenced by photooxidation of GC doublet, Nakatani, K.; Sando, S.; Yoshida, K.; Saito, I. Bioorg. Med. Chem. Lett., 2001, 11, 335-337)

Other universal bases can have the following formulas:

wherein:

Q is N or CR⁴⁴;

Q′ is N or CR⁴⁵;

Q″ is N or CR⁴⁷;

Q′″ is N or CR⁴⁹;

Q^(iv) is N or CR⁵⁰;

R⁴⁴ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b), or NR^(b)R^(c), C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, or when taken together with R⁴⁵ forms —OCH₂O—;

R⁴⁵ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b), or NR^(b)R^(c), C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, or when taken together with R⁴⁴ or R⁴⁶ forms —OCH₂O—;

R⁴⁶ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b), or NR^(b)R^(c), C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, or when taken together with R⁴⁵ or R⁴⁷ forms —OCH₂O—;

R⁴⁷ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b), or NR^(b)R^(c), C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, or when taken together with R⁴⁶ or R⁴⁸ forms —OCH2O—;

R⁴⁸ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b), or NR^(b)R^(c), C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, or when taken together with R⁴⁷ forms —OCH2O—;

R⁴⁹ R⁵⁰, R⁵¹, R⁵², R⁵³, R⁵⁴, R⁵⁷, R⁵⁸, R⁵⁹, R⁶⁰, R⁶¹, R⁶², R⁶³, R⁶⁴, R⁶⁵, R⁶⁶, R⁶⁷, R⁶⁸, R⁶⁹, R⁷⁰, R⁷¹, and R⁷² are each independently selected from hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b), or NR^(b)R^(c), C₁-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷, or NC(O)R^(o);

R⁵⁵ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b), or NR^(b)R^(c), C₁-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷, or NC(O)R^(o), or when taken together with R⁵⁶ forms a fused aromatic ring which may be optionally substituted;

R⁵⁶ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b), or NR^(b)R^(c), C₁-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷, or NC(O)R^(o), or when taken together with R⁵⁵ forms a fused aromatic ring which may be optionally substituted;

R¹⁷ is halo, NH₂, NHR^(b), or NR^(b)R^(c);

R^(b) is C₁-C₆ alkyl or a nitrogen protecting group;

R^(c) is C₁-C₆ alkyl; and

R^(o) is alkyl optionally substituted with halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b), or NR^(b)R^(c), C₁-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷, or NC(O)R^(o).

Examples of universal bases include:

In one aspect, the invention features methods of producing iRNA agents, e.g., sRNA agents, e.g. an sRNA agent described herein, having the ability to mediate RNAi. These iRNA agents can be formulated for administration to a subject.

In another aspect, the invention features a method of administering an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, to a subject (e.g., a human subject). The method includes administering a unit dose of the iRNA agent, e.g., a sRNA agent, e.g., double stranded sRNA agent that (a) the double-stranded part is 19-25 nucleotides (nt) long, preferably 21-23 nt, (b) is complementary to a target RNA (e.g., an endogenous or pathogen target RNA), and, optionally, (c) includes at least one 3′ overhang 1-5 nucleotide long. In one embodiment, the unit dose is less than 1.4 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, and less than 200 nmole of RNA agent (e.g. about 4.4×10¹⁶ copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of RNA agent per kg of bodyweight.

The defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the target RNA. The unit dose, for example, can be administered by injection (e.g., intravenous or intramuscular), an inhaled dose, or a topical application. Particularly preferred dosages are less than 2, 1, or 0.1 mg/kg of body weight.

In a preferred embodiment, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time.

In one embodiment, the effective dose is administered with other traditional therapeutic modalities. In one embodiment, the subject has a viral infection and the modality is an antiviral agent other than an iRNA agent, e.g., other than a double-stranded iRNA agent, or sRNA agent. In another embodiment, the subject has atherosclerosis and the effective dose of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, is administered in combination with, e.g., after surgical intervention, e.g., angioplasty.

In one embodiment, a subject is administered an initial dose and one or more maintenance doses of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). The maintenance dose or doses are generally lower than the initial dose, e.g., one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 μg to 1.4 mg/kg of body weight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. The maintenance doses are preferably administered no more than once every 5, 10, or 30 days.

In one embodiment, the iRNA agent pharmaceutical composition includes a plurality of iRNA agent species. In another embodiment, the iRNA agent species has sequences that are non-overlapping and non-adjacent to another species with respect to a naturally occurring target sequence. In another embodiment, the plurality of iRNA agent species is specific for different naturally occurring target genes. In another embodiment, the iRNA agent is allele specific.

The inventors have discovered that iRNA agents described herein can be administered to mammals, particularly large mammals such as nonhuman primates or humans in a number of ways.

In one embodiment, the administration of the iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, composition is parenteral, e.g. intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser that delivers a metered dose. Selected modes of delivery are discussed in more detail below.

The invention provides methods, compositions, and kits, for rectal administration or delivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes a an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of a iRNA agent described herein, e.g., a iRNA agent having a double stranded region of less than 40, and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered rectally, e.g., introduced through the rectum into the lower or upper colon. This approach is particularly useful in the treatment of, inflammatory disorders, disorders characterized by unwanted cell proliferation, e.g., polyps, or colon cancer.

In some embodiments the medication is delivered to a site in the colon by introducing a dispensing device, e.g., a flexible, camera-guided device similar to that used for inspection of the colon or removal of polyps, which includes means for delivery of the medication.

In one embodiment, the rectal administration of the iRNA agent is by means of an enema. The iRNA agent of the enema can be dissolved in a saline or buffered solution.

In another embodiment, the rectal administration is by means of a suppository. The suppository can include other ingredients, e.g., an excipient, e.g., cocoa butter or hydropropylmethylcellulose.

The invention also provides methods, compositions, and kits for oral delivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of a iRNA described herein, e.g., a iRNA agent having a double stranded region of less than 40 and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered orally.

Oral administration can be in the form of tablets, capsules, gel capsules, lozenges, troches or liquid syrups. In a preferred embodiment the composition is applied topically to a surface of the oral cavity.

The invention also provides methods, compositions, and kits for buccal delivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of iRNA agent having a double stranded region of less than 40 and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered to the buccal cavity. The medication can be sprayed into the buccal cavity or applied directly, e.g., in a liquid, solid, or gel form to a surface in the buccal cavity. This administration is particularly desirable for the treatment of inflammations of the buccal cavity, e.g., the gums or tongue, e.g., in one embodiment, the buccal administration is by spraying into the cavity, e.g., without inhalation, from a dispenser, e.g., a metered dose spray dispenser that dispenses the pharmaceutical composition and a propellant.

The invention also provides methods, compositions, and kits for ocular delivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of a iRNA agent described herein, e.g., a sRNA agent having a double stranded region of less than 40 and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered to ocular tissue.

The medications can be applied to the surface of the eye or nearby tissue, e.g., the inside of the eyelid. It can be applied topically, e.g., by spraying, in drops, as an eyewash, or an ointment. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser that delivers a metered dose.

The medication can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or structure.

Ocular treatment is particularly desirable for treating inflammation of the eye or nearby tissue.

The invention also provides methods, compositions, and kits for delivery of iRNA agents described herein to or through the skin.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of a iRNA agent described herein, e.g., a sRNA agent having a double stranded region of less than 40 and preferably less than 30 nucleotides and one or two 1-3 nucleotide single strand 3′ overhangs can be administered directly to the skin.

The medication can be applied topically or delivered in a layer of the skin, e.g., by the use of a microneedle or a battery of microneedles which penetrate into the skin, but preferably not into the underlying muscle tissue.

In one embodiment, the administration of the iRNA agent composition is topical. In another embodiment, topical administration delivers the composition to the dermis or epidermis of a subject. In other embodiments the topical administration is in the form of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids or powders. A composition for topical administration can be formulated as a liposome, micelle, emulsion, or other lipophilic molecular assembly.

In another embodiment, the transdermal administration is applied with at least one penetration enhancer. In other embodiments, the penetration can be enhanced with iontophoresis, phonophoresis, and sonophoresis. In another aspect, the invention provides methods, compositions, devices, and kits for pulmonary delivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of iRNA agent, e.g., a sRNA agent having a double stranded region of less than 40, preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered to the pulmonary system. Pulmonary administration can be achieved by inhalation or by the introduction of a delivery device into the pulmonary system, e.g., by introducing a delivery device which can dispense the medication.

The preferred method of pulmonary delivery is by inhalation. The medication can be provided in a dispenser which delivers the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered dose of medication. The subject, or another person, can administer the medication.

Pulmonary delivery is effective not only for disorders which directly affect pulmonary tissue, but also for disorders which affect other tissue.

iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or aerosol for pulmonary delivery.

In another aspect, the invention provides methods, compositions, devices, and kits for nasal delivery of iRNA agents described herein. Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of iRNA agent, e.g., a sRNA agent having a double stranded region of less than 40 and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered nasally. Nasal administration can be achieved by introduction of a delivery device into the nose, e.g., by introducing a delivery device which can dispense the medication.

The preferred method of nasal delivery is by spray, aerosol, liquid, e.g., by drops, of by topical administration to a surface of the nasal cavity. The medication can be provided in a dispenser which delivery of the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered dose of medication. The subject, or another person, can administer the medication.

Nasal delivery is effective not only for disorders which directly affect nasal tissue, but also for disorders which affect other tissue

iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or for nasal delivery.

In another embodiment, the iRNA agent is packaged in a viral natural capsid or in a chemically or enzymatically produced artificial capsid or structure derived therefrom.

In one aspect, of the invention, the dosage of a pharmaceutical composition including a iRNA agent is administered in order to alleviate the symptoms of a disease state, e.g., cancer or a cardiovascular disease.

In another aspect, gene expression in a subject is modulated by administering a pharmaceutical composition including a iRNA agent. In other embodiments, a subject is treated with the pharmaceutical composition by any of the methods mentioned above. In another embodiment, the subject has cancer.

An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition can be administered as a liposome. For example, the composition can be prepared by a method that includes: (1) contacting a iRNA agent with an amphipathic cationic lipid conjugate in the presence of a detergent; and (2) removing the detergent to form a iRNA agent and cationic lipid complex. In one embodiment, the detergent is cholate, deoxycholate, lauryl sarcosine, octanoyl sucrose, CHAPS (3-[(3-cholamidopropyl)-di-methylamine]-2-hydroxyl-1-propane), novel-θ-D-glucopyranoside, lauryl dimethylamine oxide, or octylglucoside. The iRNA agent can be an sRNA agent. The method can include preparing a composition that includes a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

In another aspect, a subject is treated by administering a defined amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent) composition that is in a powdered form. In one embodiment, the powder is a collection of microparticles. In one embodiment, the powder is a collection of crystalline particles. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

In one aspect, a subject is treated by administering a defined amount of a iRNA agent composition that is prepared by a method that includes spray-drying, i.e. atomizing a liquid solution, emulsion, or suspension, immediately exposing the droplets to a drying gas, and collecting the resulting porous powder particles. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

In one aspect, the iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), is provided in a powdered, crystallized or other finely divided form, with or without a carrier, e.g., a micro- or nano-particle suitable for inhalation or other pulmonary delivery. In one embodiment, this includes providing an aerosol preparation, e.g., an aerosolized spray-dried composition. The aerosol composition can be provided in and/or dispensed by a metered dose delivery device.

In another aspect, a subject is treated for a condition treatable by inhalation. In one embodiment, this method includes aerosolizing a spray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition and inhaling the aerosolized composition. The iRNA agent can be an sRNA. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

In another aspect, the invention features a method of treating a subject that includes: administering a composition including an effective/defined amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), wherein the composition is prepared by a method that includes spray-drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques

In another aspect, the invention features a method that includes: evaluating a parameter related to the abundance of a transcript in a cell of a subject; comparing the evaluated parameter to a reference value; and if the evaluated parameter has a preselected relationship to the reference value (e.g., it is greater), administering a iRNA agent (or a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes a iRNA agent or precursor thereof) to the subject. In one embodiment, the iRNA agent includes a sequence that is complementary to the evaluated transcript. For example, the parameter can be a direct measure of transcript levels, a measure of a protein level, a disease or disorder symptom or characterization (e.g., rate of cell proliferation and/or tumor mass, viral load,)

In another aspect, the invention features a method that includes: administering a first amount of a composition that comprises an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) to a subject, wherein the iRNA agent includes a strand substantially complementary to a target nucleic acid; evaluating an activity associated with a protein encoded by the target nucleic acid; wherein the evaluation is used to determine if a second amount should be administered. In a preferred embodiment the method includes administering a second amount of the composition, wherein the timing of administration or dosage of the second amount is a function of the evaluating. The method can include other features described herein.

In another aspect, the invention features a method of administering a source of a double-stranded iRNA agent (ds iRNA agent) to a subject. The method includes administering or implanting a source of a ds iRNA agent, e.g., a sRNA agent, that (a) includes a double-stranded region that is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to a target RNA (e.g., an endogenous RNA or a pathogen RNA), and, optionally, (c) includes at least one 3′ overhang 1-5 nt long. In one embodiment, the source releases ds iRNA agent over time, e.g. the source is a controlled or a slow release source, e.g., a microparticle that gradually releases the ds iRNA agent. In another embodiment, the source is a pump, e.g., a pump that includes a sensor or a pump that can release one or more unit doses.

In one aspect, the invention features a pharmaceutical composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) including a nucleotide sequence complementary to a target RNA, e.g., substantially and/or exactly complementary. The target RNA can be a transcript of an endogenous human gene. In one embodiment, the iRNA agent (a) is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and, optionally, (c) includes at least one 3′ overhang 1-5 nt long. In one embodiment, the pharmaceutical composition can be an emulsion, microemulsion, cream, jelly, or liposome.

In one example the pharmaceutical composition includes an iRNA agent mixed with a topical delivery agent. The topical delivery agent can be a plurality of microscopic vesicles. The microscopic vesicles can be liposomes. In a preferred embodiment the liposomes are cationic liposomes.

In another aspect, the pharmaceutical composition includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) admixed with a topical penetration enhancer. In one embodiment, the topical penetration enhancer is a fatty acid. The fatty acid can be arachidonic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₁₀ alkyl ester, monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.

In another embodiment, the topical penetration enhancer is a bile salt. The bile salt can be cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate, polyoxyethylene-9-lauryl ether or a pharmaceutically acceptable salt thereof.

In another embodiment, the penetration enhancer is a chelating agent. The chelating agent can be EDTA, citric acid, a salicylate, a N-acyl derivative of collagen, laureth-9, an N-amino acyl derivative of a beta-diketone or a mixture thereof.

In another embodiment, the penetration enhancer is a surfactant, e.g., an ionic or nonionic surfactant. The surfactant can be sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, a perfluorochemical emulsion or mixture thereof.

In another embodiment, the penetration enhancer can be selected from a group consisting of unsaturated cyclic ureas, 1-alkyl-alkones, 1-alkenylazacyclo-alakanones, steroidal anti-inflammatory agents and mixtures thereof. In yet another embodiment the penetration enhancer can be a glycol, a pyrrol, an azone, or a terpenes.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a form suitable for oral delivery. In one embodiment, oral delivery can be used to deliver an iRNA agent composition to a cell or a region of the gastro-intestinal tract, e.g., small intestine, colon (e.g., to treat a colon cancer), and so forth. The oral delivery form can be tablets, capsules or gel capsules. In one embodiment, the iRNA agent of the pharmaceutical composition modulates expression of a cellular adhesion protein, modulates a rate of cellular proliferation, or has biological activity against eukaryotic pathogens or retroviruses. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In a preferred embodiment the enteric material is a coating. The coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methylcellulose phthalate or cellulose acetate phthalate.

In another embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer. The penetration enhancer can be a bile salt or a fatty acid. The bile salt can be ursodeoxycholic acid, chenodeoxycholic acid, and salts thereof. The fatty acid can be capric acid, lauric acid, and salts thereof.

In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example the excipient is polyethyleneglycol. In another example the excipient is precirol.

In another embodiment, the oral dosage form of the pharmaceutical composition includes a plasticizer. The plasticizer can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethyl citrate.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent and a delivery vehicle. In one embodiment, the iRNA agent is (a) is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and, optionally, (c) includes at least one 3′ overhang 1-5 nucleotides long.

In one embodiment, the delivery vehicle can deliver an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) to a cell by a topical route of administration. The delivery vehicle can be microscopic vesicles. In one example the microscopic vesicles are liposomes. In a preferred embodiment the liposomes are cationic liposomes. In another example the microscopic vesicles are micelles.

In one aspect, the invention features a method for making a pharmaceutical composition, the method including: (1) contacting an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent) with a amphipathic cationic lipid conjugate in the presence of a detergent; and (2) removing the detergent to form a iRNA agent and cationic lipid complex.

In another aspect, the invention features a pharmaceutical composition produced by a method including: (1) contacting an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent) with a amphipathic cationic lipid conjugate in the presence of a detergent; and (2) removing the detergent to form a iRNA agent and cationic lipid complex. In one embodiment, the detergent is cholate, deoxycholate, lauryl sarcosine, octanoyl sucrose, CHAPS (3-[(3-cholamidopropyl)-di-methylamine]-2-hydroxyl-1-propane), novel-θ-D-glucopyranoside, lauryl dimethylamine oxide, or octylglucoside. In another embodiment, the amphipathic cationic lipid conjugate is biodegradable. In yet another embodiment the pharmaceutical composition includes a targeting ligand.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in an injectable dosage form. In one embodiment, the injectable dosage form of the pharmaceutical composition includes sterile aqueous solutions or dispersions and sterile powders. In a preferred embodiment the sterile solution can include a diluent such as water; saline solution; fixed oils, polyethylene glycols, glycerin, or propylene glycol.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in oral dosage form. In one embodiment, the oral dosage form is selected from the group consisting of tablets, capsules and gel capsules. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In a preferred embodiment the enteric material is a coating. The coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methyl cellulose phthalate or cellulose acetate phthalate. In one embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer, e.g., a penetration enhancer described herein.

In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example the excipient is polyethyleneglycol. In another example the excipient is precirol.

In another embodiment, the oral dosage form of the pharmaceutical composition includes a plasticizer. The plasticizer can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethyl citrate.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a rectal dosage form. In one embodiment, the rectal dosage form is an enema. In another embodiment, the rectal dosage form is a suppository.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a vaginal dosage form. In one embodiment, the vaginal dosage form is a suppository. In another embodiment, the vaginal dosage form is a foam, cream, or gel.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a pulmonary or nasal dosage form. In one embodiment, the iRNA agent is incorporated into a particle, e.g., a macroparticle, e.g., a microsphere. The particle can be produced by spray drying, lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination thereof. The microsphere can be formulated as a suspension, a powder, or an implantable solid.

In one aspect, the invention features a spray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition suitable for inhalation by a subject, including: (a) a therapeutically effective amount of a iRNA agent suitable for treating a condition in the subject by inhalation; (b) a pharmaceutically acceptable excipient selected from the group consisting of carbohydrates and amino acids; and (c) optionally, a dispersibility-enhancing amount of a physiologically-acceptable, water-soluble polypeptide.

In one embodiment, the excipient is a carbohydrate. The carbohydrate can be selected from the group consisting of monosaccharides, disaccharides, trisaccharides, and polysaccharides. In a preferred embodiment the carbohydrate is a monosaccharide selected from the group consisting of dextrose, galactose, mannitol, D-mannose, sorbitol, and sorbose. In another preferred embodiment the carbohydrate is a disaccharide selected from the group consisting of lactose, maltose, sucrose, and trehalose.

In another embodiment, the excipient is an amino acid. In one embodiment, the amino acid is a hydrophobic amino acid. In a preferred embodiment the hydrophobic amino acid is selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. In yet another embodiment the amino acid is a polar amino acid. In a preferred embodiment the amino acid is selected from the group consisting of arginine, histidine, lysine, cysteine, glycine, glutamine, serine, threonine, tyrosine, aspartic acid and glutamic acid.

In one embodiment, the dispersibility-enhancing polypeptide is selected from the group consisting of human serum albumin, α-lactalbumin, trypsinogen, and polyalanine.

In one embodiment, the spray-dried iRNA agent composition includes particles having a mass median diameter (MMD) of less than 10 microns. In another embodiment, the spray-dried iRNA agent composition includes particles having a mass median diameter of less than 5 microns. In yet another embodiment the spray-dried iRNA agent composition includes particles having a mass median aerodynamic diameter (MMAD) of less than 5 microns.

In certain other aspects, the invention provides kits that include a suitable container containing a pharmaceutical formulation of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). In certain embodiments the individual components of the pharmaceutical formulation may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation separately in two or more containers, e.g., one container for an iRNA agent preparation, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device.

In another aspect, the invention features a device, e.g., an implantable device, wherein the device can dispense or administer a composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), e.g., a iRNA agent that silences an endogenous transcript. In one embodiment, the device is coated with the composition. In another embodiment the iRNA agent is disposed within the device. In another embodiment, the device includes a mechanism to dispense a unit dose of the composition. In other embodiments the device releases the composition continuously, e.g., by diffusion. Exemplary devices include stents, catheters, pumps, artificial organs or organ components (e.g., artificial heart, a heart valve, etc.), and sutures.

As used herein, the term “crystalline” describes a solid having the structure or characteristics of a crystal, i.e., particles of three-dimensional structure in which the plane faces intersect at definite angles and in which there is a regular internal structure. The compositions of the invention may have different crystalline forms. Crystalline forms can be prepared by a variety of methods, including, for example, spray drying.

As used herein, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound of the invention and a target RNA molecule. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed. The non-target sequences typically differ by at least 5 nucleotides.

In one embodiment, an iRNA agent is “sufficiently complementary” to a target RNA, e.g., a target mRNA, such that the iRNA agent silences production of protein encoded by the target mRNA. In another embodiment, the iRNA agent is “exactly complementary” to a target RNA, e.g., the target RNA and the iRNA agent anneal, preferably to form a hybrid made exclusively of Watson-Crick basepairs in the region of exact complementarity. A “sufficiently complementary” target RNA can include an internal region (e.g., of at least 10 nucleotides) that is exactly complementary to a target RNA. Moreover, in some embodiments, the iRNA agent specifically discriminates a single-nucleotide difference. In this case, the iRNA agent only mediates RNAi if exact complementary is found in the region (e.g., within 7 nucleotides of) the single-nucleotide difference.

As used herein, the term “oligonucleotide” refers to a nucleic acid molecule (RNA or DNA) preferably of length less than 100, 200, 300, or 400 nucleotides.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The materials, methods, and examples are illustrative only and not intended to be limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, useful methods and materials are described below. Other features and advantages of the invention will be apparent from the accompanying drawings and description, and from the claims. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference. In case of conflict, the present specification, including definitions, will control.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a structural representation of base pairing in psuedocomplementary siRNA².

FIG. 2 is a schematic representation of dual targeting siRNAs (SEQ ID NOs 3952, 3953, 3954, and 3955 respectively) designed to target the HCV genome.

FIG. 3 is a schematic representation of pseudocomplementary, bifunctional siRNAs (SEQ ID NOs 3956, 3957, 3958, and 4087 respectively) designed to target the HCV genome.

FIG. 4 is a general synthetic scheme for incorporation of RRMS monomers into an oligonucleotide.

FIG. 5 is a table of representative RRMS carriers. Panel 1 shows pyrroline-based RRMSs; panel 2 shows 3-hydroxyproline-based RRMSs; panel 3 shows piperidine-based RRMSs; panel 4 shows morpholine and piperazine-based RRMSs; and panel 5 shows decalin-based RRMSs. R1 is succinate or phosphoramidate and R2 is H or a conjugate ligand.

FIG. 6A is a graph depicting levels of luciferase mRNA in livers of CMV-Luc mice (Xanogen) following intervenous injection (iv) of buffer or siRNA into the tail vein. Each bar represents data from one mouse. RNA levels were quantified by QuantiGene Assay (Genospectra, Inc.; Fremont, Calif.)). The Y axis represents chemiluminescence values in counts per second (CPS).

FIG. 6B is a graph depicting levels of luciferase mRNA in livers of CMV-Luc mice (Xanogen). The values are averaged from the data depicted in FIG. 6A.

FIG. 7 is a graph depicting the pharmacokinetics of cholesterol-conjugated and unconjugated siRNA. The diamonds represent the amount of unconjugated ³³P-labeled siRNA (ALN-3000) in mouse plasma over time; the squares represent the amount of cholesterol-conjugated ³³P-labeled siRNA (ALN-3001) in mouse plasma over time. “L1163” is equivalent to ALN3000; “L1163Chol” is equivalent to ALN-3001.

DETAILED DESCRIPTION

Double-stranded (dsRNA) directs the sequence-specific silencing of mRNA through a process known as RNA interference (RNAi). The process occurs in a wide variety of organisms, including mammals and other vertebrates.

It has been demonstrated that 21-23 nt fragments of dsRNA are sequence-specific mediators of RNA silencing, e.g., by causing RNA degradation. While not wishing to be bound by theory, it may be that a molecular signal, which may be merely the specific length of the fragments, present in these 21-23 nt fragments recruits cellular factors that mediate RNAi. Described herein are methods for preparing and administering these 21-23 nt fragments, and other iRNAs agents, and their use for specifically inactivating gene function. The use of iRNAs agents (or recombinantly produced or chemically synthesized oligonucleotides of the same or similar nature) enables the targeting of specific mRNAs for silencing in mammalian cells. In addition, longer dsRNA agent fragments can also be used, e.g., as described below.

Although, in mammalian cells, long dsRNAs can induce the interferon response which is frequently deleterious, sRNAs do not trigger the interferon response, at least not to an extent that is deleterious to the cell and host. In particular, the length of the iRNA agent strands in an sRNA agent can be less than 31, 30, 28, 25, or 23 nt, e.g., sufficiently short to avoid inducing a deleterious interferon response. Thus, the administration of a composition of sRNA agent (e.g., formulated as described herein) to a mammalian cell can be used to silence expression of a target gene while circumventing the interferon response. Further, use of a discrete species of iRNA agent can be used to selectively target one allele of a target gene, e.g., in a subject heterozygous for the allele.

Moreover, in one embodiment, a mammalian cell is treated with an iRNA agent that disrupts a component of the interferon response, e.g., double stranded RNA (dsRNA)-activated protein kinase PKR. Such a cell can be treated with a second iRNA agent that includes a sequence complementary to a target RNA and that has a length that might otherwise trigger the interferon response.

In a typical embodiment, the subject is a mammal such as a cow, horse, mouse, rat, dog, pig, goat, or a primate. The subject can be a dairy mammal (e.g., a cow, or goat) or other farmed animal (e.g., a chicken, turkey, sheep, pig, fish, shrimp). In a much preferred embodiment, the subject is a human, e.g., a normal individual or an individual that has, is diagnosed with, or is predicted to have a disease or disorder.

Further, because iRNA agent mediated silencing persists for several days after administering the iRNA agent composition, in many instances, it is possible to administer the composition with a frequency of less than once per day, or, for some instances, only once for the entire therapeutic regimen. For example, treatment of some cancer cells may be mediated by a single bolus administration, whereas a chronic viral infection may require regular administration, e.g., once per week or once per month.

A number of exemplary routes of delivery are described that can be used to administer an iRNA agent to a subject. In addition, the iRNA agent can be formulated according to an exemplary method described herein.

iRNA Agent Structure

Described herein are isolated iRNA agents, e.g., RNA molecules, (double-stranded; single-stranded) that mediate RNAi. The iRNA agents preferably mediate RNAi with respect to an endogenous gene of a subject or to a gene of a pathogen.

An “RNA agent” as used herein, is an unmodified RNA, modified RNA, or nucleoside surrogate, all of which are defined herein (see, e.g., the section below entitled RNA Agents). While numerous modified RNAs and nucleoside surrogates are described, preferred examples include those which have greater resistance to nuclease degradation than do unmodified RNAs. Preferred examples include those which have a 2′ sugar modification, a modification in a single strand overhang, preferably a 3′ single strand overhang, or, particularly if single stranded, a 5′ modification which includes one or more phosphate groups or one or more analogs of a phosphate group.

An “iRNA agent” as used herein, is an RNA agent which can, or which can be cleaved into an RNA agent which can, down regulate the expression of a target gene, preferably an endogenous or pathogen target RNA. While not wishing to be bound by theory, an iRNA agent may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mRNA sometimes referred to in the art as RNAi, or pre-transcriptional or pre-translational mechanisms. An iRNA agent can include a single strand or can include more than one strands, e.g., it can be a double stranded iRNA agent. If the iRNA agent is a single strand it is particularly preferred that it include a 5′ modification which includes one or more phosphate groups or one or more analogs of a phosphate group.

The iRNA agent should include a region of sufficient homology to the target gene, and be of sufficient length in terms of nucleotides, such that the iRNA agent, or a fragment thereof, can mediate down regulation of the target gene. (For ease of exposition the term nucleotide or ribonucleotide is sometimes used herein in reference to one or more monomeric subunits of an RNA agent. It will be understood herein that the usage of the term “ribonucleotide” or “nucleotide”, herein can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety at one or more positions.) Thus, the iRNA agent is or includes a region which is at least partially, and in some embodiments fully, complementary to the target RNA. It is not necessary that there be perfect complementarity between the iRNA agent and the target, but the correspondence must be sufficient to enable the iRNA agent, or a cleavage product thereof, to direct sequence specific silencing, e.g., by RNAi cleavage of the target RNA, e.g., mRNA.

Complementarity, or degree of homology with the target strand, is most critical in the antisense strand. While perfect complementarity, particularly in the antisense strand, is often desired some embodiments can include, particularly in the antisense strand, one or more but preferably 6, 5, 4, 3, 2, or fewer mismatches (with respect to the target RNA). The mismatches, particularly in the antisense strand, are most tolerated in the terminal regions and if present are preferably in a terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides of the 5′ and/or 3′ terminus. The sense strand need only be sufficiently complementary with the antisense strand to maintain the over all double strand character of the molecule.

As discussed elsewhere herein, an iRNA agent will often be modified or include nucleoside surrogates in addition to the RRMS. Single stranded regions of an iRNA agent will often be modified or include nucleoside surrogates, e.g., the unpaired region or regions of a hairpin structure, e.g., a region which links two complementary regions, can have modifications or nucleoside surrogates. Modification to stabilize one or more 3′- or 5′-terminus of an iRNA agent, e.g., against exonucleases, or to favor the antisense sRNA agent to enter into RISC are also favored. Modifications can include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents that come as phosphoramidites and that have another DMT-protected hydroxyl group, allowing multiple couplings during RNA synthesis.

iRNA agents include: molecules that are long enough to trigger the interferon response (which can be cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363-366) and enter a RISC (RNAi-induced silencing complex)); and, molecules which are sufficiently short that they do not trigger the interferon response (which molecules can also be cleaved by Dicer and/or enter a RISC), e.g., molecules which are of a size which allows entry into a RISC, e.g., molecules which resemble Dicer-cleavage products. Molecules that are short enough that they do not trigger an interferon response are termed sRNA agents or shorter iRNA agents herein. “sRNA agent or shorter iRNA agent” as used herein, refers to an iRNA agent, e.g., a double stranded RNA agent or single strand agent, that is sufficiently short that it does not induce a deleterious interferon response in a human cell, e.g., it has a duplexed region of less than 60 but preferably less than 50, 40, or 30 nucleotide pairs. The sRNA agent, or a cleavage product thereof, can down regulate a target gene, e.g., by inducing RNAi with respect to a target RNA, preferably an endogenous or pathogen target RNA.

Each strand of an sRNA agent can be equal to or less than 30, 25, 24, 23, 22, 21, or 20 nucleotides in length. The strand is preferably at least 19 nucleotides in length. For example, each strand can be between 21 and 25 nucleotides in length. Preferred sRNA agents have a duplex region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, and one or more overhangs, preferably one or two 3′ overhangs, of 2-3 nucleotides.

In addition to homology to target RNA and the ability to down regulate a target gene, an iRNA agent will preferably have one or more of the following properties:

(1) it will be of the Formula 1, 2, 3, or 4 set out in the RNA Agent section below;

(2) if single stranded it will have a 5′ modification which includes one or more phosphate groups or one or more analogs of a phosphate group;

(3) it will, despite modifications, even to a very large number, or all of the nucleosides, have an antisense strand that can present bases (or modified bases) in the proper three dimensional framework so as to be able to form correct base pairing and form a duplex structure with a homologous target RNA which is sufficient to allow down regulation of the target, e.g., by cleavage of the target RNA;

(4) it will, despite modifications, even to a very large number, or all of the nucleosides, still have “RNA-like” properties, i.e., it will possess the overall structural, chemical and physical properties of an RNA molecule, even though not exclusively, or even partly, of ribonucleotide-based content. For example, an iRNA agent can contain, e.g., a sense and/or an antisense strand in which all of the nucleotide sugars contain e.g., 2′ fluoro in place of 2′ hydroxyl. This deoxyribonucleotide-containing agent can still be expected to exhibit RNA-like properties. While not wishing to be bound by theory, the electronegative fluorine prefers an axial orientation when attached to the C2′ position of ribose. This spatial preference of fluorine can, in turn, force the sugars to adopt a C_(3′)-endo pucker. This is the same puckering mode as observed in RNA molecules and gives rise to the RNA-characteristic A-family-type helix. Further, since fluorine is a good hydrogen bond acceptor, it can participate in the same hydrogen bonding interactions with water molecules that are known to stabilize RNA structures. (Generally, it is preferred that a modified moiety at the 2′ sugar position will be able to enter into H-bonding which is more characteristic of the OH moiety of a ribonucleotide than the H moiety of a deoxyribonucleotide. A preferred iRNA agent will: exhibit a C_(3′)-endo pucker in all, or at least 50, 75, 80, 85, 90, or 95% of its sugars; exhibit a C_(3′)-endo pucker in a sufficient amount of its sugars that it can give rise to a the RNA-characteristic A-family-type helix; will have no more than 20, 10, 5, 4, 3, 2, or 1 sugar which is not a C_(3′)-endo pucker structure. These limitations are particularly preferably in the antisense strand;

(5) regardless of the nature of the modification, and even though the RNA agent can contain deoxynucleotides or modified deoxynucleotides, particularly in overhang or other single strand regions, it is preferred that DNA molecules, or any molecule in which more than 50, 60, or 70% of the nucleotides in the molecule, or more than 50, 60, or 70% of the nucleotides in a duplexed region are deoxyribonucleotides, or modified deoxyribonucleotides which are deoxy at the 2′ position, are excluded from the definition of RNA agent.

A “single strand iRNA agent” as used herein, is an iRNA agent which is made up of a single molecule. It may include a duplexed region, formed by intra-strand pairing, e.g., it may be, or include, a hairpin or pan-handle structure. Single strand iRNA agents are preferably antisense with regard to the target molecule. In preferred embodiments single strand iRNA agents are 5′ phosphorylated or include a phosphoryl analog at the 5′ prime terminus. 5′-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)2(O)—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)-5′); 5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′); 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-). (These modifications can also be used with the antisense strand of a double stranded iRNA.)

A single strand iRNA agent should be sufficiently long that it can enter the RISC and participate in RISC mediated cleavage of a target mRNA. A single strand iRNA agent is at least 14, and more preferably at least 15, 20, 25, 29, 35, 40, or 50 nucleotides in length. It is preferably less than 200, 100, or 60 nucleotides in length.

Hairpin iRNA agents will have a duplex region equal to or at least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region will preferably be equal to or less than 200, 100, or 50, in length. Preferred ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length. The hairpin will preferably have a single strand overhang or terminal unpaired region, preferably the 3′, and preferably of the antisense side of the hairpin. Preferred overhangs are 2-3 nucleotides in length.

A “double stranded (ds) iRNA agent” as used herein, is an iRNA agent which includes more than one, and preferably two, strands in which interchain hybridization can form a region of duplex structure.

The antisense strand of a double stranded iRNA agent should be equal to or at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. It should be equal to or less than 200, 100, or 50, nucleotides in length. Preferred ranges are 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.

The sense strand of a double stranded iRNA agent should be equal to or at least 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. It should be equal to or less than 200, 100, or 50, nucleotides in length. Preferred ranges are 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.

The double strand portion of a double stranded iRNA agent should be equal to or at least, 14, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotide pairs in length. It should be equal to or less than 200, 100, or 50, nucleotides pairs in length. Preferred ranges are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length.

In many embodiments, the ds iRNA agent is sufficiently large that it can be cleaved by an endogenous molecule, e.g., by Dicer, to produce smaller ds iRNA agents, e.g., sRNAs agents

It may be desirable to modify one or both of the antisense and sense strands of a double strand iRNA agent. In some cases they will have the same modification or the same class of modification but in other cases the sense and antisense strand will have different modifications, e.g., in some cases it is desirable to modify only the sense strand. It may be desirable to modify only the sense strand, e.g., to inactivate it, e.g., the sense strand can be modified in order to inactivate the sense strand and prevent formation of an active sRNA/protein or RISC. This can be accomplished by a modification which prevents 5′-phosphorylation of the sense strand, e.g., by modification with a 5′-O-methyl ribonucleotide (see Nykänen et al., (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309-321.) Other modifications which prevent phosphorylation can also be used, e.g., simply substituting the 5′-OH by H rather than 0-Me. Alternatively, a large bulky group may be added to the 5′-phosphate turning it into a phosphodiester linkage, though this may be less desirable as phosphodiesterases can cleave such a linkage and release a functional sRNA 5′-end. Antisense strand modifications include 5′ phosphorylation as well as any of the other 5′ modifications discussed herein, particularly the 5′ modifications discussed above in the section on single stranded iRNA molecules.

It is preferred that the sense and antisense strands be chosen such that the ds iRNA agent includes a single strand or unpaired region at one or both ends of the molecule. Thus, a ds iRNA agent contains sense and antisense strands, preferable paired to contain an overhang, e.g., one or two 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3 nucleotides. Most embodiments will have a 3′ overhang. Preferred sRNA agents will have single-stranded overhangs, preferably 3′ overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. 5′ ends are preferably phosphorylated.

Preferred lengths for the duplexed region is between 15 and 30, most preferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., in the sRNA agent range discussed above. sRNA agents can resemble in length and structure the natural Dicer processed products from long dsRNAs. Embodiments in which the two strands of the sRNA agent are linked, e.g., covalently linked are also included. Hairpin, or other single strand structures which provide the required double stranded region, and preferably a 3′ overhang are also within the invention.

The isolated iRNA agents described herein, including ds iRNA agents and sRNA agents can mediate silencing of a target RNA, e.g., mRNA, e.g., a transcript of a gene that encodes a protein. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene. In general, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs, can also be targeted.

As used herein, the phrase “mediates RNAi” refers to the ability to silence, in a sequence specific manner, a target RNA. While not wishing to be bound by theory, it is believed that silencing uses the RNAi machinery or process and a guide RNA, e.g., an sRNA agent of 21 to 23 nucleotides.

As used herein, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound of the invention and a target RNA molecule. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed. The non-target sequences typically differ by at least 5 nucleotides.

In one embodiment, an iRNA agent is “sufficiently complementary” to a target RNA, e.g., a target mRNA, such that the iRNA agent silences production of protein encoded by the target mRNA. In another embodiment, the iRNA agent is “exactly complementary” (excluding the RRMS containing subunit(s)) to a target RNA, e.g., the target RNA and the iRNA agent anneal, preferably to form a hybrid made exclusively of Watson-Crick basepairs in the region of exact complementarity. A “sufficiently complementary” target RNA can include an internal region (e.g., of at least 10 nucleotides) that is exactly complementary to a target RNA. Moreover, in some embodiments, the iRNA agent specifically discriminates a single-nucleotide difference. In this case, the iRNA agent only mediates RNAi if exact complementary is found in the region (e.g., within 7 nucleotides of) the single-nucleotide difference.

As used herein, the term “oligonucleotide” refers to a nucleic acid molecule (RNA or DNA) preferably of length less than 100, 200, 300, or 400 nucleotides.

RNA agents discussed herein include otherwise unmodified RNA as well as RNA which have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al., (1994) Summary: the modified nucleosides of RNA, Nucleic Acids Res. 22: 2183-2196. Such rare or unusual RNAs, often termed modified RNAs (apparently because the are typically the result of a post transcriptionally modification) are within the term unmodified RNA, as used herein. Modified RNA as used herein refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, preferably different from that which occurs in the human body. While they are referred to as modified “RNAs,” they will of course, because of the modification, include molecules which are not RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to the presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone. Examples of all of the above are discussed herein.

Much of the discussion below refers to single strand molecules. In many embodiments of the invention a double stranded iRNA agent, e.g., a partially double stranded iRNA agent, is required or preferred. Thus, it is understood that that double stranded structures (e.g. where two separate molecules are contacted to form the double stranded region or where the double stranded region is formed by intramolecular pairing (e.g., a hairpin structure)) made of the single stranded structures described below are within the invention. Preferred lengths are described elsewhere herein.

As nucleic acids are polymers of subunits or monomers, many of the modifications described below occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or the a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many, and in fact in most cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal regions, e.g. at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of an RNA or may only occur in a single strand region of an RNA. E.g., a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal regions, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5′ end or ends can be phosphorylated.

In some embodiments it is particularly preferred, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′ or 5′ overhang will be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ OH group of the ribose sugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine, instead of ribonucleotides, and modifications in the phosphate group, e.g., phosphothioate modifications. Overhangs need not be homologous with the target sequence.

Modifications and nucleotide surrogates are discussed below.

The scaffold presented above in Formula 1 represents a portion of a ribonucleic acid. The basic components are the ribose sugar, the base, the terminal phosphates, and phosphate internucleotide linkers. Where the bases are naturally occurring bases, e.g., adenine, uracil, guanine or cytosine, the sugars are the unmodified 2′ hydroxyl ribose sugar (as depicted) and W, X, Y, and Z are all O, Formula 1 represents a naturally occurring unmodified oligoribonucleotide.

Unmodified oligoribonucleotides may be less than optimal in some applications, e.g., unmodified oligoribonucleotides can be prone to degradation by e.g., cellular nucleases. Nucleases can hydrolyze nucleic acid phosphodiester bonds. However, chemical modifications to one or more of the above RNA components can confer improved properties, and, e.g., can render oligoribonucleotides more stable to nucleases. Unmodified oligoribonucleotides may also be less than optimal in terms of offering tethering points for attaching ligands or other moieties to an iRNA agent.

Modified nucleic acids and nucleotide surrogates can include one or more of:

(i) alteration, e.g., replacement, of one or both of the non-linking (X and Y) phosphate oxygens and/or of one or more of the linking (W and Z) phosphate oxygens (When the phosphate is in the terminal position, one of the positions W or Z will not link the phosphate to an additional element in a naturally occurring ribonucleic acid. However, for simplicity of terminology, except where otherwise noted, the W position at the 5′ end of a nucleic acid and the terminal Z position at the 3′ end of a nucleic acid, are within the term “linking phosphate oxygens” as used herein.);

(ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar, or wholesale replacement of the ribose sugar with a structure other than ribose, e.g., as described herein;

(iii) wholesale replacement of the phosphate moiety (bracket I) with “dephospho” linkers;

(iv) modification or replacement of a naturally occurring base;

(v) replacement or modification of the ribose-phosphate backbone (bracket II);

(vi) modification of the 3′ end or 5′ end of the RNA, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, e.g. a fluorescently labeled moiety, to either the 3′ or 5′ end of RNA.

The terms replacement, modification, alteration, and the like, as used in this context, do not imply any process limitation, e.g., modification does not mean that one must start with a reference or naturally occurring ribonucleic acid and modify it to produce a modified ribonucleic acid bur rather modified simply indicates a difference from a naturally occurring molecule.

It is understood that the actual electronic structure of some chemical entities cannot be adequately represented by only one canonical form (i.e. Lewis structure). While not wishing to be bound by theory, the actual structure can instead be some hybrid or weighted average of two or more canonical forms, known collectively as resonance forms or structures. Resonance structures are not discrete chemical entities and exist only on paper. They differ from one another only in the placement or “localization” of the bonding and nonbonding electrons for a particular chemical entity. It can be possible for one resonance structure to contribute to a greater extent to the hybrid than the others. Thus, the written and graphical descriptions of the embodiments of the present invention are made in terms of what the art recognizes as the predominant resonance form for a particular species. For example, any phosphoroamidate (replacement of a nonlinking oxygen with nitrogen) would be represented by X=O and Y=N in the above figure.

Specific modifications are discussed in more detail below.

The Phosphate Group

The phosphate group is a negatively charged species. The charge is distributed equally over the two non-linking oxygen atoms (i.e., X and Y in Formula 1 above). However, the phosphate group can be modified by replacing one of the oxygens with a different substituent. One result of this modification to RNA phosphate backbones can be increased resistance of the oligoribonucleotide to nucleolytic breakdown. Thus while not wishing to be bound by theory, it can be desirable in some embodiments to introduce alterations which result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.

Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. Unlike the situation where only one of X or Y is altered, the phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligoribonucleotides diastereomers. Diastereomer formation can result in a preparation in which the individual diastereomers exhibit varying resistance to nucleases. Further, the hybridization affinity of RNA containing chiral phosphate groups can be lower relative to the corresponding unmodified RNA species. Thus, while not wishing to be bound by theory, modifications to both X and Y which eliminate the chiral center, e.g. phosphorodithioate formation, may be desirable in that they cannot produce diastereomer mixtures. Thus, X can be any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl). Thus Y can be any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl). Replacement of X and/or Y with sulfur is preferred.

The phosphate linker can also be modified by replacement of a linking oxygen (i.e., W or Z in Formula 1) with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at a terminal oxygen (position W (3′) or position Z (5′). Replacement of W with carbon or Z with nitrogen is preferred.

Candidate agents can be evaluated for suitability as described below.

The Sugar Group A modified RNA can include modification of all or some of the sugar groups of the ribonucleic acid. E.g., the 2′ hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents. While not being bound by theory, enhanced stability is expected since the hydroxyl can no longer be deprotonated to form a 2′ alkoxide ion. The 2′ alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom. Again, while not wishing to be bound by theory, it can be desirable to some embodiments to introduce alterations in which alkoxide formation at the 2′ position is not possible.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked” nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by a methylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE (AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino) and aminoalkoxy, O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino). It is noteworthy that oligonucleotides containing only the methoxyethyl group (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nuclease stabilities comparable to those modified with the robust phosphorothioate modification.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, which are of particular relevance to the overhang portions of partially ds RNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino), —NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality. Preferred substitutents are 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl, 2′-C-allyl, and 2′-fluoro.

The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified RNA can include nucleotides containing e.g., arabinose, as the sugar.

Modified RNA's can also include “abasic” sugars, which lack a nucleobase at C-1′. These abasic sugars can also be further contain modifications at one or more of the constituent sugar atoms.

To maximize nuclease resistance, the 2′ modifications can be used in combination with one or more phosphate linker modifications (e.g., phosphorothioate). The so-called “chimeric” oligonucleotides are those that contain two or more different modifications.

The modification can also entail the wholesale replacement of a ribose structure with another entity at one or more sites in the iRNA agent. These modifications are described in section entitled Ribose Replacements for RRMSs.

Candidate modifications can be evaluated as described below.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containing connectors (cf. Bracket I in Formula 1 above). While not wishing to be bound by theory, it is believed that since the charged phosphodiester group is the reaction center in nucleolytic degradation, its replacement with neutral structural mimics should impart enhanced nuclease stability. Again, while not wishing to be bound by theory, it can be desirable, in some embodiment, to introduce alterations in which the charged phosphate group is replaced by a neutral moiety.

Examples of moieties which can replace the phosphate group include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. Preferred replacements include the methylenecarbonylamino and methylenemethylimino groups.

Candidate modifications can be evaluated as described below.

Replacement of Ribophosphate Backbone

Oligonucleotide-mimicking scaffolds can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates (see Bracket II of Formula 1 above). While not wishing to be bound by theory, it is believed that the absence of a repetitively charged backbone diminishes binding to proteins that recognize polyanions (e.g. nucleases). Again, while not wishing to be bound by theory, it can be desirable in some embodiment, to introduce alterations in which the bases are tethered by a neutral surrogate backbone.

Examples include the mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. A preferred surrogate is a PNA surrogate.

Candidate modifications can be evaluated as described below.

Terminal Modifications

The 3′ and 5′ ends of an oligonucleotide can be modified. Such modifications can be at the 3′ end, 5′ end or both ends of the molecule. They can include modification or replacement of an entire terminal phosphate or of one or more of the atoms of the phosphate group. E.g., the 3′ and 5′ ends of an oligonucleotide can be conjugated to other functional molecular entities such as labeling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron or ester). The functional molecular entities can be attached to the sugar through a phosphate group and/or a spacer. The terminal atom of the spacer can connect to or replace the linking atom of the phosphate group or the C-3′ or C-5′ O, N, S or C group of the sugar. Alternatively, the spacer can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs). These spacers or linkers can include e.g., —(CH₂)_(n)—, —(CH₂)_(n)N—, —(CH₂)_(n)O—, —(CH₂)_(n)S—, O(CH₂CH₂O)_(n)CH₂CH₂OH (e.g., n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents. When a spacer/phosphate-functional molecular entity-spacer/phosphate array is interposed between two strands of iRNA agents, this array can substitute for a hairpin RNA loop in a hairpin-type RNA agent. The 3′ end can be an —OH group. While not wishing to be bound by theory, it is believed that conjugation of certain moieties can improve transport, hybridization, and specificity properties. Again, while not wishing to be bound by theory, it may be desirable to introduce terminal alterations that improve nuclease resistance. Other examples of terminal modifications include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic carriers (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles).

Terminal modifications can be added for a number of reasons, including as discussed elsewhere herein to modulate activity or to modulate resistance to degradation. Terminal modifications useful for modulating activity include modification of the 5′ end with phosphate or phosphate analogs. E.g., in preferred embodiments iRNA agents, especially antisense strands, are 5′ phosphorylated or include a phosphoryl analog at the 5′ prime terminus. 5′-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 4′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′); 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-).

Terminal modifications useful for increasing resistance to degradation include

Terminal modifications can also be useful for monitoring distribution, and in such cases the preferred groups to be added include fluorophores, e.g., fluorscein or an Alexa dye, e.g., Alexa 488. Terminal modifications can also be useful for enhancing uptake, useful modifications for this include cholesterol. Terminal modifications can also be useful for cross-linking an RNA agent to another moiety; modifications useful for this include mitomycin C.

Candidate modifications can be evaluated as described below.

The Bases

Adenine, guanine, cytosine and uracil are the most common bases found in RNA. These bases can be modified or replaced to provide RNA's having improved properties. E.g., nuclease resistant oligoribonucleotides can be prepared with these bases or with synthetic and natural nucleobases (e.g., inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) and any one of the above modifications. Alternatively, substituted or modified analogs of any of the above bases, e.g., “unusual bases” and “universal bases,” can be employed. Examples include without limitation 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases. Further purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613.

Generally, base changes are less preferred for promoting stability, but they can be useful for other reasons, e.g., some, e.g., 2,6-diaminopurine and 2 amino purine, are fluorescent. Modified bases can reduce target specificity. This should be taken into consideration in the design of iRNA agents.

Candidate modifications can be evaluated as described below.

Evaluation of Candidate RNA's

One can evaluate a candidate RNA agent, e.g., a modified RNA, for a selected property by exposing the agent or modified molecule and a control molecule to the appropriate conditions and evaluating for the presence of the selected property. For example, resistance to a degradent can be evaluated as follows. A candidate modified RNA (and preferably a control molecule, usually the unmodified form) can be exposed to degradative conditions, e.g., exposed to a milieu, which includes a degradative agent, e.g., a nuclease. E.g., one can use a biological sample, e.g., one that is similar to a milieu, which might be encountered, in therapeutic use, e.g., blood or a cellular fraction, e.g., a cell-free homogenate or disrupted cells. The candidate and control could then be evaluated for resistance to degradation by any of a number of approaches. For example, the candidate and control could be labeled, preferably prior to exposure, with, e.g., a radioactive or enzymatic label, or a fluorescent label, such as Cy3 or Cy5. Control and modified RNA's can be incubated with the degradative agent, and optionally a control, e.g., an inactivated, e.g., heat inactivated, degradative agent. A physical parameter, e.g., size, of the modified and control molecules are then determined. They can be determined by a physical method, e.g., by polyacrylamide gel electrophoresis or a sizing column, to assess whether the molecule has maintained its original length, or assessed functionally. Alternatively, Northern blot analysis can be used to assay the length of an unlabeled modified molecule.

A functional assay can also be used to evaluate the candidate agent. A functional assay can be applied initially or after an earlier non-functional assay, (e.g., assay for resistance to degradation) to determine if the modification alters the ability of the molecule to silence gene expression. For example, a cell, e.g., a mammalian cell, such as a mouse or human cell, can be co-transfected with a plasmid expressing a fluorescent protein, e.g., GFP, and a candidate RNA agent homologous to the transcript encoding the fluorescent protein (see, e.g., WO 00/44914). For example, a modified dsRNA homologous to the GFP mRNA can be assayed for the ability to inhibit GFP expression by monitoring for a decrease in cell fluorescence, as compared to a control cell, in which the transfection did not include the candidate dsRNA, e.g., controls with no agent added and/or controls with a non-modified RNA added. Efficacy of the candidate agent on gene expression can be assessed by comparing cell fluorescence in the presence of the modified and unmodified dsRNA agents.

In an alternative functional assay, a candidate dsRNA agent homologous to an endogenous mouse gene, preferably a maternally expressed gene, such as c-mos, can be injected into an immature mouse oocyte to assess the ability of the agent to inhibit gene expression in vivo (see, e.g., WO 01/36646). A phenotype of the oocyte, e.g., the ability to maintain arrest in metaphase II, can be monitored as an indicator that the agent is inhibiting expression. For example, cleavage of c-mos mRNA by a dsRNA agent would cause the oocyte to exit metaphase arrest and initiate parthenogenetic development (Colledge et al. Nature 370: 65-68, 1994; Hashimoto et al. Nature, 370:68-71, 1994). The effect of the modified agent on target RNA levels can be verified by Northern blot to assay for a decrease in the level of target mRNA, or by Western blot to assay for a decrease in the level of target protein, as compared to a negative control. Controls can include cells in which with no agent is added and/or cells in which a non-modified RNA is added.

REFERENCES General References

The oligoribonucleotides and oligoribonucleosides used in accordance with this invention may be with solid phase synthesis, see for example “Oligonucleotide synthesis, a practical approach”, Ed. M. J. Gait, IRL Press, 1984; “Oligonucleotides and Analogues, A Practical Approach”, Ed. F. Eckstein, IRL Press, 1991 (especially Chapter 1, Modern machine-aided methods of oligodeoxyribonucleotide synthesis, Chapter 2, Oligoribonucleotide synthesis, Chapter 3, 2′-O-Methyloligoribonucleotide-s: synthesis and applications, Chapter 4, Phosphorothioate oligonucleotides, Chapter 5, Synthesis of oligonucleotide phosphorodithioates, Chapter 6, Synthesis of oligo-2′-deoxyribonucleoside methylphosphonates, and. Chapter 7, Oligodeoxynucleotides containing modified bases. Other particularly useful synthetic procedures, reagents, blocking groups and reaction conditions are described in Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48, 2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49, 6123-6194, or references referred to therein.

Modification described in WO 00/44895, WO01/75164, or WO02/44321 can be used herein.

The disclosure of all publications, patents, and published patent applications listed herein are hereby incorporated by reference.

Phosphate Group References

The preparation of phosphinate oligoribonucleotides is described in U.S. Pat. No. 5,508,270. The preparation of alkyl phosphonate oligoribonucleotides is described in U.S. Pat. No. 4,469,863. The preparation of phosphoramidite oligoribonucleotides is described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878. The preparation of phosphotriester oligoribonucleotides is described in U.S. Pat. No. 5,023,243. The preparation of borano phosphate oligoribonucleotide is described in U.S. Pat. Nos. 5,130,302 and 5,177,198. The preparation of 3′-Deoxy-3′-amino phosphoramidate oligoribonucleotides is described in U.S. Pat. No. 5,476,925. 3′-Deoxy-3′-methylenephosphonate oligoribonucleotides is described in An, H, et al. J. Org. Chem. 2001, 66, 2789-2801. Preparation of sulfur bridged nucleotides is described in Sproat et al. Nucleosides Nucleotides 1988, 7, 651 and Crosstick et al. Tetrahedron Lett. 1989, 30, 4693.

Sugar Group References

Modifications to the 2′ modifications can be found in Verma, S. et al. Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein. Specific modifications to the ribose can be found in the following references: 2′-fluoro (Kawasaki et. al., J. Med. Chem., 1993, 36, 831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938), “LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310).

Replacement of the Phosphate Group References

Methylenemethylimino linked oligoribonucleosides, also identified herein as MMI linked oligoribonucleosides, methylenedimethylhydrazo linked oligoribonucleosides, also identified herein as MDH linked oligoribonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified herein as amide-3 linked oligoribonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified herein as amide-4 linked oligoribonucleosides as well as mixed backbone compounds having, as for instance, alternating MMI and PO or PS linkages can be prepared as is described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and in published PCT applications PCT/US92/04294 and PCT/US92/04305 (published as WO 92/20822 WO and 92/20823, respectively). Formacetal and thioformacetal linked oligoribonucleosides can be prepared as is described in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxide linked oligoribonucleosides can be prepared as is described in U.S. Pat. No. 5,223,618. Siloxane replacements are described in Cormier, J. F. et al. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements are described in Tittensor, J. R. J. Chem. Soc. C 1971, 1933. Carboxymethyl replacements are described in Edge, M. D. et al. J. Chem. Soc. Perkin Trans. 1 1972, 1991. Carbamate replacements are described in Stirchak, E. P. Nucleic Acids Res. 1989, 17, 6129.

Replacement of the Phosphate-Ribose Backbone References

Cyclobutyl sugar surrogate compounds can be prepared as is described in U.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate can be prepared as is described in U.S. Pat. No. 5,519,134. Morpholino sugar surrogates can be prepared as is described in U.S. Pat. Nos. 5,142,047 and 5,235,033, and other related patent disclosures. Peptide Nucleic Acids (PNAs) are known per se and can be prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. No. 5,539,083.

Terminal Modification References

Terminal modifications are described in Manoharan, M. et al. Antisense and Nucleic Acid Drug Development 12, 103-128 (2002) and references therein.

Bases References

N-2 substituted purine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,459,255. 3-Deaza purine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,457,191. 5,6-Substituted pyrimidine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,614,617. 5-Propynyl pyrimidine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,484,908. Additional references can be disclosed in the above section on base modifications.

Preferred iRNA Agents

Preferred RNA agents have the following structure (see Formula 2 below):

Referring to Formula 2 above, R¹, R², and R³ are each, independently, H, (i.e. abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.

R⁴, R⁵, and R⁶ are each, independently, OR⁸, O(CH₂CH₂O)_(m)CH₂CH₂OR⁸; O(CH₂)_(n)R⁹; O(CH₂)_(n)OR⁹, H; halo; NH₂; NHR⁸; N(R⁸)₂; NH(CH₂CH₂NH)_(m)CH₂CH₂NHR⁹; NHC(O)R⁸; cyano; mercapto, SR⁸; alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of which may be optionally substituted with halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido; or R⁴, R⁵, or R⁶ together combine with R⁷ to form an [—O—CH₂—] covalently bound bridge between the sugar 2′ and 4′ carbons.

A¹ is:

H; OH; OCH₃; W¹; an abasic nucleotide; or absent;

(a preferred A1, especially with regard to anti-sense strands, is chosen from 5′-monophosphate ((HO)₂(O)P—O-5′), 5′-diphosphate ((HO)₂(O)P—O—P(HO)(O)—O-5′), 5′-triphosphate ((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-monothiophosphate (phosphorothioate; (HO)₂(S)P—O-5′), 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)₂(O)P—S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, (OH)₂(O)P-5′-CH₂—), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH₂—), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-)).

A² is:

A³ is:

and

A⁴ is:

H; Z⁴; an inverted nucleotide; an abasic nucleotide; or absent.

W¹ is OH, (CH₂)_(n)R¹⁰, (CH₂)_(n)NHR¹⁰, (CH₂)_(n)OR¹⁰, (CH₂)_(n)SR¹⁰; O(CH₂)_(n)R¹⁰; O(CH₂)_(n)OR¹⁰, O(CH₂)_(n)NR¹⁰, O(CH₂)_(n)SR¹⁰; O(CH₂)_(n)SS(CH₂)_(n)OR¹⁰, O(CH₂)_(n)C(O)OR¹⁰, NH(CH₂)_(n)R¹⁰; NH(CH₂)_(n)NR¹⁰; NH(CH₂)_(n)OR¹⁰, NH(CH₂)_(n)SR¹⁰; S(CH₂)_(n)R¹⁰, S(CH₂)_(n)NR¹⁰, S(CH₂)_(n)OR¹⁰, S(CH₂)_(n)SR¹⁰O(CH₂CH₂O)_(m)CH₂CH₂OR¹⁰; O(CH₂CH₂O)_(m)CH₂CH₂NHR¹⁰, NH(CH₂CH₂NH)_(m)CH₂CH₂NHR¹⁰; Q-R¹⁰, O-Q-R¹⁰N-Q-R¹⁰, S-Q-R¹⁰ or —O—. W⁴ is O, CH₂, NH, or S.

X¹, X², X³, and X⁴ are each, independently, O or S.

Y¹, Y², Y³, and Y⁴ are each, independently, OH, O⁻, OR⁸, S, Se, BH₃ ⁻, H, NHR⁹, N(R⁹)₂ alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted.

Z¹, Z², and Z³ are each independently O, CH₂, NH, or S. Z⁴ is OH, (CH₂)_(n)R¹⁰, (CH₂)_(n)NHR¹⁰, (CH₂)_(n)OR¹⁰, (CH₂)_(n)SR¹⁰; O(CH₂)_(n)R¹⁰; O(CH₂OR¹⁰, O(CH₂)_(n)NR¹⁰, O(CH₂)_(n)SR¹⁰, O(CH₂)_(n)SS(CH₂)_(n)OR¹⁰, O(CH₂)_(n)C(O)OR¹⁰; NH(CH₂)_(n)R¹⁰; NH(CH₂)_(n)NR¹⁰; NH(CH₂)_(n)OR¹⁰, NH(CH₂)_(n)SR¹⁰; S(CH₂)_(n)R¹⁰, S(CH₂)_(n)NR¹⁰, S(CH₂)_(n)OR¹⁰, S(CH₂)_(n)SR¹⁰O(CH₂CH₂O)_(m)CH₂CH₂OR¹⁰, O(CH₂CH₂O)_(m)CH₂CH₂NHR¹⁰, NH(CH₂CH₂NH)_(m)CH₂CH₂NHR¹⁰; Q-R¹⁰, O-Q-R¹⁰N-Q-R¹⁰, Q-S-R¹⁰.

x is 5-100, chosen to comply with a length for an RNA agent described herein.

R⁷ is H; or is together combined with R⁴, R⁵, or R⁶ to form an [—O—CH₂—] covalently bound bridge between the sugar 2′ and 4′ carbons.

R⁸ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, amino acid, or sugar; R⁹ is NH₂, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid; and R¹⁰ is H; fluorophore (pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes); sulfur, silicon, boron or ester protecting group; intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4,texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipohilic carriers (cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino; alkyl, cycloalkyl, aryl, aralkyl, heteroaryl; radiolabelled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles); or an RNA agent. m is 0-1,000,000, and n is 0-20. Q is a spacer selected from the group consisting of abasic sugar, amide, carboxy, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, biotin or fluorescein reagents.

Preferred RNA agents in which the entire phosphate group has been replaced have the following structure (see Formula 3 below):

Referring to Formula 3, A¹⁰-A⁴⁰ is L-G-L; A¹⁰ and/or A⁴⁰ may be absent, in which L is a linker, wherein one or both L may be present or absent and is selected from the group consisting of CH₂(CH₂)_(g); N(CH₂)_(g); O(CH₂)_(g); S(CH₂)_(g). G is a functional group selected from the group consisting of siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

R¹⁰, R²⁰, and R³⁰ are each, independently, H, (i.e. abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.

R⁴⁰, R⁵⁰, and R⁶⁰ are each, independently, OR⁸, O(CH₂CH₂O)_(m)CH₂CH₂OR⁸; O(CH₂)_(n)R⁹; O(CH₂)_(n)OR⁹, H; halo; NH₂; NHR⁸; N(R⁸)₂; NH(CH₂CH₂NH)_(m)CH₂CH₂R⁹; NHC(O)R⁸; cyano; mercapto, SR⁷; alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of which may be optionally substituted with halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups; or R⁴⁰, R⁵⁰, or R⁶⁰ together combine with R⁷⁰ to form an [—O—CH₂—] covalently bound bridge between the sugar 2′ and 4′ carbons.

x is 5-100 or chosen to comply with a length for an RNA agent described herein.

R⁷⁰ is H; or is together combined with R⁴⁰, R⁵⁰, or R⁶⁰ to form an [—O—CH₂—] covalently bound bridge between the sugar 2′ and 4′ carbons.

R⁸ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, amino acid, or sugar; and R⁹ is NH₂, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid. m is 0-1,000,000, n is 0-20, and g is 0-2.

Preferred nucleoside surrogates have the following structure (see Formula 4 below): SLR¹⁰⁰-(M-SLR²⁰⁰)_(x)-M-SLR³⁰⁰  FORMULA 4

S is a nucleoside surrogate selected from the group consisting of mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid. L is a linker and is selected from the group consisting of CH₂(CH₂)_(g); N(CH₂)_(g); O(CH₂)_(g); S(CH₂)_(g); —C(O)(CH₂)_(n)— or may be absent. M is an amide bond; sulfonamide; sulfinate; phosphate group; modified phosphate group as described herein; or may be absent.

R¹⁰⁰, R²⁰⁰, and R³⁰⁰ are each, independently, H (i.e., abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, 7-deazaguanine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted 1,2,4-triazoles, 2-pyridinones, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.

x is 5-100, or chosen to comply with a length for an RNA agent described herein; and g is 0-2.

Nuclease Resistant Monomers

In one aspect, the invention features a nuclease resistant monomer, or a an iRNA agent which incorporates a nuclease resistant monomer (NMR), such as those described herein and those described in copending, co-owned U.S. Provisional Application Ser. No. 60/469,612, filed on May 9, 2003, which is hereby incorporated by reference.

In addition, the invention includes iRNA agents having a NMR and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a NMR.

An iRNA agent can include monomers which have been modified so as to inhibit degradation, e.g., by nucleases, e.g., endonucleases or exonucleases, found in the body of a subject. These monomers are referred to herein as NRM's, or nuclease resistance promoting monomers or modifications. In many cases these modifications will modulate other properties of the iRNA agent as well, e.g., the ability to interact with a protein, e.g., a transport protein, e.g., serum albumin, or a member of the RISC (RNA-induced Silencing Complex), or the ability of the first and second sequences to form a duplex with one another or to form a duplex with another sequence, e.g., a target molecule.

While not wishing to be bound by theory, it is believed that modifications of the sugar, base, and/or phosphate backbone in an iRNA agent can enhance endonuclease and exonuclease resistance, and can enhance interactions with transporter proteins and one or more of the functional components of the RISC complex. Preferred modifications are those that increase exonuclease and endonuclease resistance and thus prolong the halflife of the iRNA agent prior to interaction with the RISC complex, but at the same time do not render the iRNA agent resistant to endonuclease activity in the RISC complex. Again, while not wishing to be bound by any theory, it is believed that placement of the modifications at or near the 3′ and/or 5′ end of antisense strands can result in iRNA agents that meet the preferred nuclease resistance criteria delineated above. Again, still while not wishing to be bound by any theory, it is believed that placement of the modifications at e.g., the middle of a sense strand can result in iRNA agents that are relatively less likely to undergo off-targeting.

Modifications described herein can be incorporated into any double-stranded RNA and RNA-like molecule described herein, e.g., an iRNA agent. An iRNA agent may include a duplex comprising a hybridized sense and antisense strand, in which the antisense strand and/or the sense strand may include one or more of the modifications described herein. The anti sense strand may include modifications at the 3′ end and/or the 5′ end and/or at one or more positions that occur 1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. The sense strand may include modifications at the 3′ end and/or the 5′ end and/or at any one of the intervening positions between the two ends of the strand. The iRNA agent may also include a duplex comprising two hybridized antisense strands. The first and/or the second antisense strand may include one or more of the modifications described herein. Thus, one and/or both antisense strands may include modifications at the 3′ end and/or the 5′ end and/or at one or more positions that occur 1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. Particular configurations are discussed below.

Modifications that can be useful for producing iRNA agents that meet the preferred nuclease resistance criteria delineated above can include one or more of the following chemical and/or stereochemical modifications of the sugar, base, and/or phosphate backbone:

(i) chiral (S_(P)) thioates. Thus, preferred NRM's include nucleotide dimers with an enriched or pure for a particular chiral form of a modified phosphate group containing a heteroatom at the nonbridging position, e.g., Sp or Rp, at the position X, where this is the position normally occupied by the oxygen. The atom at X can also be S, Se, Nr₂, or Br₃. When X is S, enriched or chirally pure Sp linkage is preferred. Enriched means at least 70, 80, 90, 95, or 99% of the preferred form. Such NRM's are discussed in more detail below;

(ii) attachment of one or more cationic groups to the sugar, base, and/or the phosphorus atom of a phosphate or modified phosphate backbone moiety. Thus, preferred NRM's include monomers at the terminal position derivitized at a cationic group. As the 5′ end of an antisense sequence should have a terminal —OH or phosphate group this NRM is preferrably not used at th 5′ end of an anti-sense sequence. The group should be attached at a position on the base which minimizes interference with H bond formation and hybridization, e.g., away form the face which interacts with the complementary base on the other strand, e.g, at the 5′ position of a pyrimidine or a 7-position of a purine. These are discussed in more detail below;

(iii) nonphosphate linkages at the termini. Thus, preferred NRM's include Non-phosphate linkages, e.g., a linkage of 4 atoms which confers greater resistance to cleavage than does a phosphate bond. Examples include 3′ CH2-NCH₃—O—CH2-5′ and 3′ CH2-NH—(O═)—CH2-5′;

(iv) 3′-bridging thiophosphates and 5′-bridging thiophosphates. Thus, preferred NRM's can included these structures;

(v) L-RNA, 2′-5′ linkages, inverted linkages, a-nucleosides. Thus, other preferred NRM's include: L nucleosides and dimeric nucleotides derived from L-nucleosides; 2′-5′ phosphate, non-phosphate and modified phosphate linkages (e.g., thiophosphates, phosphoramidates and boronophosphates); dimers having inverted linkages, e.g., 3′-3′ or 5′-5′ linkages; monomers having an alpha linkage at the 1′ site on the sugar, e.g., the structures described herein having an alpha linkage;

(vi) conjugate groups. Thus, preferred NRM's can include e.g., a targeting moiety or a conjugated ligand described herein conjugated with the monomer, e.g., through the sugar, base, or backbone;

(vi) abasic linkages. Thus, preferred NRM's can include an abasic monomer, e.g., an abasic monomer as described herein (e.g., a nucleobaseless monomer); an aromatic or heterocyclic or polyheterocyclic aromatic monomer as described herein.; and

(vii) 5′-phosphonates and 5′-phosphate prodrugs. Thus, preferred NRM's include monomers, preferably at the terminal position, e.g., the 5′ position, in which one or more atoms of the phosphate group is derivatized with a protecting group, which protecting group or groups, are removed as a result of the action of a component in the subject's body, e.g, a carboxyesterase or an enzyme present in the subject's body. E.g., a phosphate prodrug in which a carboxy esterase cleaves the protected molecule resulting in the production of a thioate anion which attacks a carbon adjacent to the O of a phosphate and resulting in the production of an unprotected phosphate.

One or more different NRM modifications can be introduced into an iRNA agent or into a sequence of an iRNA agent. An NRM modification can be used more than once in a sequence or in an iRNA agent. As some NRM's interfere with hybridization the total number incorporated, should be such that acceptable levels of iRNA agent duplex formation are maintained.

In some embodiments NRM modifications are introduced into the terminal the cleavage site or in the cleavage region of a sequence (a sense strand or sequence) which does not target a desired sequence or gene in the subject. This can reduce off-target silencing.

Chiral S_(P) Thioates

A modification can include the alteration, e.g., replacement, of one or both of the non-linking (X and Y) phosphate oxygens and/or of one or more of the linking (W and Z) phosphate oxygens. Formula X below depicts a phosphate moiety linking two sugar/sugar surrogate-base moities, SB₁ and SB₂.

In certain embodiments, one of the non-linking phosphate oxygens in the phosphate backbone moiety (X and Y) can be replaced by any one of the following: S, Se, BR₃ (R is hydrogen, alkyl, aryl, etc.), C (i.e., an alkyl group, an aryl group, etc.), H, NR₂ (R is hydrogen, alkyl, aryl, etc.), or OR (R is alkyl or aryl). The phosphorus atom in an unmodified phosphate group is achiral. However, replacement of one of the non-linking oxygens with one of the above atoms or groups of atoms renders the phosphorus atom chiral; in other words a phosphorus atom in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphorus atom can possess either the “R” configuration (herein R_(P)) or the “S” configuration (herein S_(P)). Thus if 60% of a population of stereogenic phosphorus atoms have the R_(P) configuration, then the remaining 40% of the population of stereogenic phosphorus atoms have the S_(P) configuration.

In some embodiments, iRNA agents, having phosphate groups in which a phosphate non-linking oxygen has been replaced by another atom or group of atoms, may contain a population of stereogenic phosphorus atoms in which at least about 50% of these atoms (e.g., at least about 60% of these atoms, at least about 70% of these atoms, at least about 80% of these atoms, at least about 90% of these atoms, at least about 95% of these atoms, at least about 98% of these atoms, at least about 99% of these atoms) have the S_(P) configuration. Alternatively, iRNA agents having phosphate groups in which a phosphate non-linking oxygen has been replaced by another atom or group of atoms may contain a population of stereogenic phosphorus atoms in which at least about 50% of these atoms (e.g., at least about 60% of these atoms, at least about 70% of these atoms, at least about 80% of these atoms, at least about 90% of these atoms, at least about 95% of these atoms, at least about 98% of these atoms, at least about 99% of these atoms) have the R_(P) configuration. In other embodiments, the population of stereogenic phosphorus atoms may have the S_(P) configuration and may be substantially free of stereogenic phosphorus atoms having the R_(P) configuration. In still other embodiments, the population of stereogenic phosphorus atoms may have the R_(P) configuration and may be substantially free of stereogenic phosphorus atoms having the S_(P) configuration. As used herein, the phrase “substantially free of stereogenic phosphorus atoms having the R_(P) configuration” means that moieties containing stereogenic phosphorus atoms having the R_(P) configuration cannot be detected by conventional methods known in the art (chiral HPLC, ¹H NMR analysis using chiral shift reagents, etc.). As used herein, the phrase “substantially free of stereogenic phosphorus atoms having the S_(P) configuration” means that moieties containing stereogenic phosphorus atoms having the S_(P) configuration cannot be detected by conventional methods known in the art (chiral HPLC, ¹H NMR analysis using chiral shift reagents, etc.).

In a preferred embodiment, modified iRNA agents contain a phosphorothioate group, i.e., a phosphate groups in which a phosphate non-linking oxygen has been replaced by a sulfur atom. In an especially preferred embodiment, the population of phosphorothioate stereogenic phosphorus atoms may have the S_(P) configuration and be substantially free of stereogenic phosphorus atoms having the R_(P) configuration.

Phosphorothioates may be incorporated into iRNA agents using dimers e.g., formulas X-1 and X-2. The former can be used to introduce phosphorothioate

at the 3′ end of a strand, while the latter can be used to introduce this modification at the 5′ end or at a position that occurs e.g., 1, 2, 3, 4, 5, or 6 nucleotides from either end of the strand. In the above formulas, Y can be 2-cyanoethoxy, W and Z can be O, R_(2′) can be, e.g., a substituent that can impart the C-3 endo configuration to the sugar (e.g., OH, F, OCH₃), DMT is dimethoxytrityl, and “BASE” can be a natural, unusual, or a universal base.

X-1 and X-2 can be prepared using chiral reagents or directing groups that can result in phosphorothioate-containing dimers having a population of stereogenic phosphorus atoms having essentially only the R_(P) configuration (i.e., being substantially free of the S_(P) configuration) or only the S_(P) configuration (i.e., being substantially free of the R_(P) configuration). Alternatively, dimers can be prepared having a population of stereogenic phosphorus atoms in which about 50% of the atoms have the R_(P) configuration and about 50% of the atoms have the S_(P) configuration. Dimers having stereogenic phosphorus atoms with the R_(P) configuration can be identified and separated from dimers having stereogenic phosphorus atoms with the S_(P) configuration using e.g., enzymatic degradation and/or conventional chromatography techniques.

Cationic Groups

Modifications can also include attachment of one or more cationic groups to the sugar, base, and/or the phosphorus atom of a phosphate or modified phosphate backbone moiety. A cationic group can be attached to any atom capable of substitution on a natural, unusual or universal base. A preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bonding interactions needed for base pairing. A cationic group can be attached e.g., through the C2′ position of a sugar or analogous position in a cyclic or acyclic sugar surrogate. Cationic groups can include e.g., protonated amino groups, derived from e.g., O-AMINE (AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); aminoalkoxy, e.g., O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino).

Nonphosphate Linkages

Modifications can also include the incorporation of nonphosphate linkages at the 5′ and/or 3′ end of a strand. Examples of nonphosphate linkages which can replace the phosphate group include methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. Preferred replacements include the methyl phosphonate and hydroxylamino groups.

3′-Bridging Thiophosphates and 5′-Bridging Thiophosphates; Locked-RNA, 2′-5′ Linkages, Inverted Linkages, α-Nucleosides; Conjugate Groups; Abasic Linkages; and 5′-Phosphonates and 5′ Phosphate Prodrugs

Referring to formula X above, modifications can include replacement of one of the bridging or linking phosphate oxygens in the phosphate backbone moiety (W and Z). Unlike the situation where only one of X or Y is altered, the phosphorus center in the phosphorodithioates is achiral which precludes the formation of iRNA agents containing a stereogenic phosphorus atom.

Modifications can also include linking two sugars via a phosphate or modified phosphate group through the 2′ position of a first sugar and the 5′ position of a second sugar. Also contemplated are inverted linkages in which both a first and second sugar are each linked through the respective 3′ positions. Modified RNA's can also include “abasic” sugars, which lack a nucleobase at C-1′. The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified iRNA agent can include nucleotides containing e.g., arabinose, as the sugar. In another subset of this modification, the natural, unusual, or universal base may have the α-configuration. Modifications can also include L-RNA.

Modifications can also include 5′-phosphonates, e.g., P(O)(O⁻)₂—X—C^(5′)-sugar (X=CH2, CF2, CHF and 5′-phosphate prodrugs, e.g., P(O)[OCH2CH2SC(O)R]₂CH₂C^(5′)-sugar. In the latter case, the prodrug groups may be decomposed via reaction first with carboxy esterases. The remaining ethyl thiolate group via intramolecular S_(N)2 displacement can depart as episulfide to afford the underivatized phosphate group.

Modification can also include the addition of conjugating groups described elsewhere herein, which are preferably attached to an iRNA agent through any amino group available for conjugation.

Nuclease resistant modifications include some which can be placed only at the terminus and others which can go at any position. Generally the modifications that can inhibit hybridization so it is preferably to use them only in terminal regions, and preferable to not use them at the cleavage site or in the cleavage region of an sequence which targets a subject sequence or gene. The can be used anywhere in a sense sequence, provided that sufficient hybridization between the two sequences of the iRNA agent is maintained. In some embodiments it is desirable to put the NRM at the cleavage site or in the cleavage region of a sequence which does not target a subject sequence or gene, as it can minimize off-target silencing.

In addition, an iRNA agent described herein can have an overhang which does not form a duplex structure with the other sequence of the iRNA agent—it is an overhang, but it does hybridize, either with itself, or with another nucleic acid, other than the other sequence of the iRNA agent.

In most cases, the nuclease-resistance promoting modifications will be distributed differently depending on whether the sequence will target a sequence in the subject (often referred to as an anti-sense sequence) or will not target a sequence in the subject (often referred to as a sense sequence). If a sequence is to target a sequence in the subject, modifications which interfere with or inhibit endonuclease cleavage should not be inserted in the region which is subject to RISC mediated cleavage, e.g., the cleavage site or the cleavage region (As described in Elbashir et al., 2001, Genes and Dev. 15: 188, hereby incorporated by reference, cleavage of the target occurs about in the middle of a 20 or 21 nt guide RNA, or about 10 or 11 nucleotides upstream of the first nucleotide which is complementary to the guide sequence. As used herein cleavage site refers to the nucleotide on either side of the cleavage site, on the target or on the iRNA agent strand which hybridizes to it. Cleavage region means an nucleotide with 1, 2, or 3 nucleotides of the cleave site, in either direction.)

Such modifications can be introduced into the terminal regions, e.g., at the terminal position or with 2, 3, 4, or 5 positions of the terminus, of a sequence which targets or a sequence which does not target a sequence in the subject.

An iRNA agent can have a first and a second strand chosen from the following:

a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end and which has a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

a first strand which does not target a sequence and which has an NRM modification at the cleavage site or in the cleavage region;

a first strand which does not target a sequence and which has an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3′ and the 5′ end; and

a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand);

a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end and which has a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

a second strand which targets a sequence and which preferably does not have an NRM modification at the cleavage site or in the cleavage region;

a second strand which targets a sequence and which does not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3′ and the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand).

An iRNA agent can also target two sequences and can have a first and second strand chosen from:

a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand);

a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end and which has a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

a first strand which targets a sequence and which preferably does not have an NRM modification at the cleavage site or in the cleavage region;

a first strand which targets a sequence and which dose not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3′ and the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand) and

a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand);

a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end and which has a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

a second strand which targets a sequence and which preferably does not have an NRM modification at the cleavage site or in the cleavage region;

a second strand which targets a sequence and which dose not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3′ and the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand).

Ribose Mimics

In one aspect, the invention features a ribose mimic, or an iRNA agent which incorporates a ribose mimic, such as those described herein and those described in copending co-owned U.S. Provisional Application Ser. No. 60/454,962, filed on Mar. 13, 2003, which is hereby incorporated by reference.

In addition, the invention includes iRNA agents having a ribose mimic and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a ribose mimic.

Thus, an aspect of the invention features an iRNA agent that includes a secondary hydroxyl group, which can increase efficacy and/or confer nuclease resistance to the agent. Nucleases, e.g., cellular nucleases, can hydrolyze nucleic acid phosphodiester bonds, resulting in partial or complete degradation of the nucleic acid. The secondary hydroxy group confers nuclease resistance to an iRNA agent by rendering the iRNA agent less prone to nuclease degradation relative to an iRNA which lacks the modification. While not wishing to be bound by theory, it is believed that the presence of a secondary hydroxyl group on the iRNA agent can act as a structural mimic of a 3′ ribose hydroxyl group, thereby causing it to be less susceptible to degradation.

The secondary hydroxyl group refers to an “OH” radical that is attached to a carbon atom substituted by two other carbons and a hydrogen. The secondary hydroxyl group that confers nuclease resistance as described above can be part of any acyclic carbon-containing group. The hydroxyl may also be part of any cyclic carbon-containing group, and preferably one or more of the following conditions is met (1) there is no ribose moiety between the hydroxyl group and the terminal phosphate group or (2) the hydroxyl group is not on a sugar moiety which is coupled to a base. The hydroxyl group is located at least two bonds (e.g., at least three bonds away, at least four bonds away, at least five bonds away, at least six bonds away, at least seven bonds away, at least eight bonds away, at least nine bonds away, at least ten bonds away, etc.) from the terminal phosphate group phosphorus of the iRNA agent. In preferred embodiments, there are five intervening bonds between the terminal phosphate group phosphorus and the secondary hydroxyl group.

Preferred iRNA agent delivery modules with five intervening bonds between the terminal phosphate group phosphorus and the secondary hydroxyl group have the following structure (see formula Y below):

Referring to formula Y, A is an iRNA agent, including any iRNA agent described herein. The iRNA agent may be connected directly or indirectly (e.g., through a spacer or linker) to “W” of the phosphate group. These spacers or linkers can include e.g., —(CH₂)_(n)—, —(CH₂)_(n)N—, —(CH₂)_(n)O—, —(CH₂)_(n)S—, O(CH₂CH₂O)_(n)CH₂CH₂OH (e.g., n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents.

The iRNA agents can have a terminal phosphate group that is unmodified (e.g., W, X, Y, and Z are O) or modified. In a modified phosphate group, W and Z can be independently NH, O or S; and X and Y can be independently S, Se, BH₃ ⁻, C₁-C₆ alkyl, C₆-C₁₀ aryl, H, O, O⁻, alkoxy or amino (including alkylamino, arylamino, etc.). Preferably, W, X and Z are O and Y is S.

R₁ and R₃ are each, independently, hydrogen; or C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl.

R₂ is hydrogen; C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R₂ may be taken together with R₄ or R₆ to form a ring of 5-12 atoms.

R₄ is hydrogen; C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R₄ may be taken together with R₂ or R₅ to form a ring of 5-12 atoms.

R₅ is hydrogen, C₁-C₁₀₀ alkyl optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R₅ may be taken together with R₄ to form a ring of 5-12 atoms.

R₆ is hydrogen, C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl, or, when n is 1, R₆ may be taken together with R₂ to form a ring of 6-10 atoms;

R₇ is hydrogen, C₁-C₁₀₀ alkyl, or C(O)(CH₂)_(q)C(O)NHR₉; T is hydrogen or a functional group; n and q are each independently 1-100; R₈ is C₁-C₁₀ alkyl or C₆-C₁₀ aryl; and R₉ is hydrogen, C1-C10 alkyl, C6-C10 aryl or a solid support agent.

Preferred embodiments may include one of more of the following subsets of iRNA agent delivery modules.

In one subset of RNAi agent delivery modules, A can be connected directly or indirectly through a terminal 3′ or 5′ ribose sugar carbon of the RNA agent.

In another subset of RNAi agent delivery modules, X, W, and Z are O and Y is S.

In still yet another subset of RNAi agent delivery modules, n is 1, and R₂ and R₆ are taken together to form a ring containing six atoms and R₄ and R₅ are taken together to form a ring containing six atoms. Preferably, the ring system is a trans-decalin. For example, the RNAi agent delivery module of this subset can include a compound of Formula (Y-1):

The functional group can be, for example, a targeting group (e.g., a steroid or a carbohydrate), a reporter group (e.g., a fluorophore), or a label (an isotopically labelled moiety). The targeting group can further include protein binding agents, endothelial cell targeting groups (e.g., RGD peptides and mimetics), cancer cell targeting groups (e.g., folate Vitamin B12, Biotin), bone cell targeting groups (e.g., bisphosphonates, polyglutamates, polyaspartates), multivalent mannose (for e.g., macrophage testing), lactose, galactose, N-acetyl-galactosamine, monoclonal antibodies, glycoproteins, lectins, melanotropin, or thyrotropin.

As can be appreciated by the skilled artisan, methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art. The synthesized compounds can be separated from a reaction mixture and further purified by a method such as column chromatography, high pressure liquid chromatography, or recrystallization. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

Ribose Replacement Monomer Subunits

iRNA agents can be modified in a number of ways which can optimize one or more characteristics of the iRNA agent. In one aspect, the invention features a ribose replacement monomer subunit (RRMS), or a an iRNA agent which incorporates a RRMS, such as those described herein and those described in one or more of U.S. Provisional Application Ser. No. 60/493,986, filed on Aug. 8, 2003, which is hereby incorporated by reference; U.S. Provisional Application Ser. No. 60/494,597, filed on Aug. 11, 2003, which is hereby incorporated by reference; U.S. Provisional Application Ser. No. 60/506,341, filed on Sep. 26, 2003, which is hereby incorporated by reference; and in U.S. Provisional Application Ser. No. 60/158,453, filed on Nov. 7, 2003, which is hereby incorporated by reference.

In addition, the invention includes iRNA agents having a RRMS and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a RRMS.

The ribose sugar of one or more ribonucleotide subunits of an iRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

The carriers further include (i) at least two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” as used herein refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a ligand, e.g., a targeting or delivery moiety, or a moiety which alters a physical property, e.g., lipophilicity, of an iRNA agent. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, it will include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.

Incorporation of one or more RRMSs described herein into an RNA agent, e.g., an iRNA agent, particularly when tethered to an appropriate entity, can confer one or more new properties to the RNA agent and/or alter, enhance or modulate one or more existing properties in the RNA molecule. E.g., it can alter one or more of lipophilicity or nuclease resistance. Incorporation of one or more RRMSs described herein into an iRNA agent can, particularly when the RRMS is tethered to an appropriate entity, modulate, e.g., increase, binding affinity of an iRNA agent to a target mRNA, change the geometry of the duplex form of the iRNA agent, alter distribution or target the iRNA agent to a particular part of the body, or modify the interaction with nucleic acid binding proteins (e.g., during RISC formation and strand separation).

Accordingly, in one aspect, the invention features, an iRNA agent preferably comprising a first strand and a second strand, wherein at least one subunit having a formula (R-1) is incorporated into at least one of said strands.

Referring to formula (R-1), X is N(CO)R⁷, NR⁷ or CH₂; Y is NR⁸, O, S, CR⁹R¹⁰, or absent; and Z is C¹¹R¹² or absent.

Each of R¹, R², R³, R⁴, R⁹, and R¹⁰ is, independently, H, OR^(a), OR^(b), (CH₂)_(n)OR^(a), or (CH₂)_(n)OR^(b), provided that at least one of R¹, R², R³, R⁴, R⁹, and R¹⁰ is OR^(a) or OR^(b) and that at least one of R¹, R², R³, R⁴, R⁹, and R¹⁰ is (CH₂)_(n)OR^(a), or (CH₂)_(n)OR^(b) (when the RRMS is terminal, one of R¹, R², R³, R⁴, R⁹, and R¹⁰ will include R^(a) and one will include R^(b); when the RRMS is internal, two of R¹, R², R³, R⁴, R⁹, and R¹⁰ will each include an R^(b)); further provided that preferably OR^(a) may only be present with (CH₂)_(n)OR^(b) and (CH₂)_(n)OR^(a) may only be present with OR^(b).

Each of R⁵, R⁶, R¹¹, and R¹² is, independently, H, C₁-C₆ alkyl optionally substituted with 1-3 R¹³, or C(O)NHR⁷; or R⁵ and R¹¹ together are C₃-C₈ cycloalkyl optionally substituted with R¹⁴.

R⁷ is C₁-C₂₀ alkyl substituted with NR^(c)R^(d); R⁸ is C₁-C₆ alkyl; R¹³ is hydroxy, C₁-C₄ alkoxy, or halo; and R¹⁴ is NR^(c)R⁷.

R^(a) is:

and

R^(b) is:

Each of A and C is, independently, O or S.

B is OH, O⁻, or

R^(c) is H or C1-C6 alkyl; R^(d) is H or a ligand; and n is 1-4.

In a preferred embodiment the ribose is replaced with a pyrroline scaffold, and X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is absent.

In other preferred embodiments the ribose is replaced with a piperidine scaffold, and X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CR¹¹R¹².

In other preferred embodiments the ribose is replaced with a piperazine scaffold, and X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR¹¹R¹².

In other preferred embodiments the ribose is replaced with a morpholino scaffold, and X is N(CO)R⁷ or NR⁷, Y is O, and Z is CR¹¹R¹².

In other preferred embodiments the ribose is replaced with a decalin scaffold, and X is CH₂; Y is CR⁹R¹⁰; and Z is CR¹¹R¹²; and R⁵ and R¹¹ together are C⁶ cycloalkyl.

In other preferred embodiments the ribose is replaced with a decalin/indane scaffold and, and X is CH₂; Y is CR⁹R¹⁰; and Z is CR¹¹R¹²; and R⁵ and R¹¹ together are C⁵ cycloalkyl.

In other preferred embodiments, the ribose is replaced with a hydroxyproline scaffold.

RRMSs described herein may be incorporated into any double-stranded RNA-like molecule described herein, e.g., an iRNA agent. An iRNA agent may include a duplex comprising a hybridized sense and antisense strand, in which the antisense strand and/or the sense strand may include one or more of the RRMSs described herein. An RRMS can be introduced at one or more points in one or both strands of a double-stranded iRNA agent. An RRMS can be placed at or near (within 1, 2, or 3 positions) of the 3′ or 5′ end of the sense strand or at near (within 2 or 3 positions of) the 3′ end of the antisense strand. In some embodiments it is preferred to not have an RRMS at or near (within 1, 2, or 3 positions of) the 5′ end of the antisense strand. An RRMS can be internal, and will preferably be positioned in regions not critical for antisense binding to the target.

In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand and at (or within 1, 2, or 3 positions of) the 3′ end of the sense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand and an RRMS at the 5′ end of the sense strand, in which both ligands are located at the same end of the iRNA agent.

In certain embodiments, two ligands are tethered, preferably, one on each strand and are hydrophobic moieties. While not wishing to be bound by theory, it is believed that pairing of the hydrophobic ligands can stabilize the iRNA agent via intermolecular van der Waals interactions.

In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand and an RRMS at the 5′ end of the sense strand, in which both RRMSs may share the same ligand (e.g., cholic acid) via connection of their individual tethers to separate positions on the ligand. A ligand shared between two proximal RRMSs is referred to herein as a “hairpin ligand.”

In other embodiments, an iRNA agent may have an RRMS at the 3′ end of the sense strand and an RRMS at an internal position of the sense strand. An iRNA agent may have an RRMS at an internal position of the sense strand; or may have an RRMS at an internal position of the antisense strand; or may have an RRMS at an internal position of the sense strand and an RRMS at an internal position of the antisense strand.

In preferred embodiments the iRNA agent includes a first and second sequences, which are preferably two separate molecules as opposed to two sequences located on the same strand, have sufficient complementarity to each other to hybridize (and thereby form a duplex region), e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme.

It is preferred that the first and second sequences be chosen such that the ds iRNA agent includes a single strand or unpaired region at one or both ends of the molecule. Thus, a ds iRNA agent contains first and second sequences, preferable paired to contain an overhang, e.g., one or two 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3 nucleotides. Most embodiments will have a 3′ overhang. Preferred sRNA agents will have single-stranded overhangs, preferably 3′ overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. 5′ ends are preferably phosphorylated.

An RNA agent, e.g., an iRNA agent, containing a preferred, but nonlimiting RRMS is presented as formula (R-2) in FIG. 4. The carrier includes two “backbone attachment points” (hydroxyl groups), a “tethering attachment point,” and a ligand, which is connected indirectly to the carrier via an intervening tether. The RRMS may be the 5′ or 3′ terminal subunit of the RNA molecule, i.e., one of the two “W” groups may be a hydroxyl group, and the other “W” group may be a chain of two or more unmodified or modified ribonucleotides. Alternatively, the RRMS may occupy an internal position, and both “W” groups may be one or more unmodified or modified ribonucleotides. More than one RRMS may be present in a RNA molecule, e.g., an iRNA agent.

The modified RNA molecule of formula (R-2) can be obtained using oligonucleotide synthetic methods known in the art. In a preferred embodiment, the modified RNA molecule of formula (II) can be prepared by incorporating one or more of the corresponding RRMS monomer compounds (RRMS monomers, see, e.g., A, B, and C in FIG. 4) into a growing sense or antisense strand, utilizing, e.g., phosphoramidite or H-phosphonate coupling strategies.

The RRMS monomers generally include two differently functionalized hydroxyl groups (OFG¹ and OFG² above), which are linked to the carrier molecule (see A in FIG. 4), and a tethering attachment point. As used herein, the term “functionalized hydroxyl group” means that the hydroxyl proton has been replaced by another substituent. As shown in representative structures B and C, one hydroxyl group (OFG¹) on the carrier is functionalized with a protecting group (PG). The other hydroxyl group (OFG²) can be functionalized with either (1) a liquid or solid phase synthesis support reagent (solid circle) directly or indirectly through a linker, L, as in B, or (2) a phosphorus-containing moiety, e.g., a phosphoramidite as in C. The tethering attachment point may be connected to a hydrogen atom, a tether, or a tethered ligand at the time that the monomer is incorporated into the growing sense or antisense strand (see R in Scheme 1). Thus, the tethered ligand can be, but need not be attached to the monomer at the time that the monomer is incorporated into the growing strand. In certain embodiments, the tether, the ligand or the tethered ligand may be linked to a “precursor” RRMS after a “precursor” RRMS monomer has been incorporated into the strand.

The (OFG¹) protecting group may be selected as desired, e.g., from T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991). The protecting group is preferably stable under amidite synthesis conditions, storage conditions, and oligonucleotide synthesis conditions. Hydroxyl groups, —OH, are nucleophilic groups (i.e., Lewis bases), which react through the oxygen with electrophiles (i.e., Lewis acids). Hydroxyl groups in which the hydrogen has been replaced with a protecting group, e.g., a triarylmethyl group or a trialkylsilyl group, are essentially unreactive as nucleophiles in displacement reactions. Thus, the protected hydroxyl group is useful in preventing e.g., homocoupling of compounds exemplified by structure C during oligonucleotide synthesis. A preferred protecting group is the dimethoxytrityl group.

When the OFG² in B includes a linker, e.g., a long organic linker, connected to a soluble or insoluble support reagent, solution or solid phase synthesis techniques can be employed to build up a chain of natural and/or modified ribonucleotides once OFG¹ is deprotected and free to react as a nucleophile with another nucleoside or monomer containing an electrophilic group (e.g., an amidite group). Alternatively, a natural or modified ribonucleotide or oligoribonucleotide chain can be coupled to monomer C via an amidite group or H-phosphonate group at OFG². Subsequent to this operation, OFG¹ can be deblocked, and the restored nucleophilic hydroxyl group can react with another nucleoside or monomer containing an electrophilic group (see FIG. 1). R′ can be substituted or unsubstituted alkyl or alkenyl. In preferred embodiments, R′ is methyl, allyl or 2-cyanoethyl. R″ may a C₁-C₁₀ alkyl group, preferably it is a branched group containing three or more carbons, e.g., isopropyl.

OFG² in B can be hydroxyl functionalized with a linker, which in turn contains a liquid or solid phase synthesis support reagent at the other linker terminus. The support reagent can be any support medium that can support the monomers described herein. The monomer can be attached to an insoluble support via a linker, L, which allows the monomer (and the growing chain) to be solubilized in the solvent in which the support is placed. The solubilized, yet immobilized, monomer can react with reagents in the surrounding solvent; unreacted reagents and soluble by-products can be readily washed away from the solid support to which the monomer or monomer-derived products is attached. Alternatively, the monomer can be attached to a soluble support moiety, e.g., polyethylene glycol (PEG) and liquid phase synthesis techniques can be used to build up the chain. Linker and support medium selection is within skill of the art. Generally the linker may be —C(O)(CH₂)_(q)C(O)—, or —C(O)(CH₂)_(q)S—, preferably, it is oxalyl, succinyl or thioglycolyl. Standard control pore glass solid phase synthesis supports can not be used in conjunction with fluoride labile 5′ silyl protecting groups because the glass is degraded by fluoride with a significant reduction in the amount of full-length product. Fluoride-stable polystyrene based supports or PEG are preferred.

Preferred carriers have the general formula (R-3) provided below. (In that structure preferred backbone attachment points can be chosen from R¹ or R²; R³ or R⁴; or R⁹ and R¹⁰ if Y is CR⁹R¹⁰ (two positions are chosen to give two backbone attachment points, e.g., R¹ and R⁴, or R⁴ and R⁹. Preferred tethering attachment points include R⁷; R⁵ or R⁶ when X is CH₂. The carriers are described below as an entity, which can be incorporated into a strand. Thus, it is understood that the structures also encompass the situations wherein one (in the case of a terminal position) or two (in the case of an internal position) of the attachment points, e.g., R¹ or R²; R³ or R⁴; or R⁹ or R¹⁰ (when Y is CR⁹R¹⁰), is connected to the phosphate, or modified phosphate, e.g., sulfur containing, backbone. E.g., one of the above-named R groups can be —CH2-, wherein one bond is connected to the carrier and one to a backbone atom, e.g., a linking oxygen or a central phosphorus atom.)

X is N(CO)R⁷, NR⁷ or CH₂; Y is NR⁸, O, S, CR⁹R¹⁰; and Z is CR¹¹R¹² or absent.

Each of R¹, R², R³, R⁴, R⁹, and R¹⁰ is, independently, H, OR^(a), or (CH₂)_(n)OR^(b), provided that at least two of R¹, R², R³, R⁴, R⁹, and R¹⁰ are OR^(a) and/or (CH₂)_(n)OR^(b).

Each of R⁵, R⁶, R¹¹, and R¹² is, independently, a ligand, H, C₁-C₆ alkyl optionally substituted with 1-3 R¹³, or C(O)NHR⁷; or R⁵ and R¹¹ together are C₃-C₈ cycloalkyl optionally substituted with R¹⁴.

R⁷ is H, a ligand, or C₁-C₂₀ alkyl substituted with NR^(c)R^(d); R⁸ is H or C₁-C₆ alkyl; R¹³ is hydroxy, C₁-C₄ alkoxy, or halo; R¹⁴ is NR^(c)R⁷; R¹⁵ is C₁-C₆ alkyl optionally substituted with cyano, or C₂-C₆ alkenyl; R¹⁶ is C₁-C₁₀ alkyl; and R¹⁷ is a liquid or solid phase support reagent.

L is —C(O)(CH₂)_(q)C(O)—, or —C(O)(CH₂)_(q)S—; R^(a) is CAr₃; R^(b) is P(O)(O⁻)H, P(OR¹⁵)N(R¹⁶)₂ or L-R¹⁷; R^(c) is H or C₁-C₆ alkyl; and R^(d) is H or a ligand.

Each Ar is, independently, C₆-C₁₀ aryl optionally substituted with C₁-C₄ alkoxy; n is 1-4; and q is 0-4.

Exemplary carriers include those in which, e.g., X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is absent; or X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CR¹¹R¹²; or X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR¹¹R¹²; or X is N(CO)R⁷ or NR⁷, Y is O, and Z is CR¹¹R¹²; or X is CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵ and R¹¹ together form C₆ cycloalkyl (H, z=2), or the indane ring system, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², R⁵ and R¹¹ together form C₅ cycloalkyl (H, z=1).

In certain embodiments, the carrier may be based on the pyrroline ring system or the 3-hydroxyproline ring system, e.g., X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is absent (D). OFG¹ is preferably attached to a primary carbon, e.g., an exocyclic alkylene

group, e.g., a methylene group, connected to one of the carbons in the five-membered ring (—CH₂OFG¹ in D). OFG² is preferably attached directly to one of the carbons in the five-membered ring (—OFG² in D). For the pyrroline-based carriers, —CH₂OFG¹ may be attached to C-2 and OFG² may be attached to C-3; or —CH₂OFG¹ may be attached to C-3 and OFG² may be attached to C-4. In certain embodiments, CH₂OFG¹ and OFG² may be geminally substituted to one of the above-referenced carbons. For the 3-hydroxyproline-based carriers, —CH₂OFG¹ may be attached to C-2 and OFG² may be attached to C-4. The pyrroline- and 3-hydroxyproline-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, CH₂OFG¹ and OFG² may be cis or trans with respect to one another in any of the pairings delineated above Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen.

In certain embodiments, the carrier may be based on the piperidine ring system (E), e.g., X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CR¹¹R¹². OFG¹ is preferably

attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group (n=1) or ethylene group (n=2), connected to one of the carbons in the six-membered ring [—(CH₂)_(n)OFG¹ in E]. OFG² is preferably attached directly to one of the carbons in the six-membered ring (—OFG² in E). —(CH₂)_(n)OFG¹ and OFG² may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, or C-4. Alternatively, —(CH₂)_(n)OFG¹ and OFG² may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., —(CH₂)_(n)OFG¹ may be attached to C-2 and OFG² may be attached to C-3; —(CH₂)_(n)OFG¹ may be attached to C-3 and OFG² may be attached to C-2; —(CH₂)_(n)OFG¹ may be attached to C-3 and OFG² may be attached to C-4; or —(CH₂)_(n)OFG¹ may be attached to C-4 and OFG² may be attached to C-3. The piperidine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, —(CH₂)_(n)OFG¹ and OFG² may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen.

In certain embodiments, the carrier may be based on the piperazine ring system (F), e.g., X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR¹¹R¹², or the morpholine ring system (G), e.g., X is N(CO)R⁷ or NR⁷, Y is O, and Z is CR¹¹R¹². OFG¹ is preferably

attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, connected to one of the carbons in the six-membered ring (—CH₂OFG¹ in F or G). OFG² is preferably attached directly to one of the carbons in the six-membered rings (—OFG² in F or G). For both F and G, —CH₂OFG¹ may be attached to C-2 and OFG² may be attached to C-3; or vice versa. In certain embodiments, CH₂OFG¹ and OFG² may be geminally substituted to one of the above-referenced carbons. The piperazine- and morpholine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, CH₂OFG¹ and OFG² may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. R′″ can be, e.g., C₁-C₆ alkyl, preferably CH₃. The tethering attachment point is preferably nitrogen in both F and G.

In certain embodiments, the carrier may be based on the decalin ring system, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵ and R¹¹ together form C₆ cycloalkyl (H, z=2), or the indane ring system, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵ and R¹¹ together form C₅ cycloalkyl (H, z=1). OFG¹ is preferably attached to a primary carbon,

e.g., an exocyclic methylene group (n=1) or ethylene group (n=2) connected to one of C-2, C-3, C-4, or C-5 [—(CH₂)_(n)OFG¹ in H]. OFG² is preferably attached directly to one of C-2, C-3, C-4, or C-5 (—OFG² in H). —(CH₂)_(n)OFG¹ and OFG² may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, C-4, or C-5. Alternatively, —(CH₂)_(n)OFG¹ and OFG² may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., —(CH₂)_(n)OFG¹ may be attached to C-2 and OFG² may be attached to C-3; —(CH₂)_(n)OFG¹ may be attached to C-3 and OFG² may be attached to C-2; —(CH₂)_(n)OFG¹ may be attached to C-3 and OFG² may be attached to C-4; or —(CH₂)_(n)OFG¹ may be attached to C-4 and OFG² may be attached to C-3; —(CH₂)_(n)OFG¹ may be attached to C-4 and OFG² may be attached to C-5; or —(CH₂)_(n)OFG¹ may be attached to C-5 and OFG² may be attached to C-4. The decalin or indane-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, —(CH₂)_(n)OFG¹ and OFG² may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. In a preferred embodiment, the substituents at C-1 and C-6 are trans with respect to one another. The tethering attachment point is preferably C-6 or C-7.

Other carriers may include those based on 3-hydroxyproline (J). Thus, —(CH₂)_(n)OFG¹ and OFG² may be cis or trans with respect to one another. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers

and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen.

Representative carriers are shown in FIG. 5.

In certain embodiments, a moiety, e.g., a ligand may be connected indirectly to the carrier via the intermediacy of an intervening tether. Tethers are connected to the carrier at the tethering attachment point (TAP) and may include any C₁-C₁₀₀ carbon-containing moiety, (e.g. C₁-C₇₅, C₁-C₅₀, C₁-C₂₀, C₁-C₁₀, C₁-C₆), preferably having at least one nitrogen atom. In preferred embodiments, the nitrogen atom forms part of a terminal amino group on the tether, which may serve as a connection point for the ligand. Preferred tethers (underlined) include TAP-(CH₂)_(n)NH₂ ; TAP-C(O)(CH₂)_(n)NH₂ ; or TAP-NR″″(CH₂)_(n)NH₂ , in which n is 1-6 and R″″ is C₁-C₆ alkyl. and R^(d) is hydrogen or a ligand. In other embodiments, the nitrogen may form part of a terminal oxyamino group, e.g., —ONH₂, or hydrazino group, —NHNH₂. The tether may optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S. Preferred tethered ligands may include, e.g., TAP-(CH₂)_(n)NH(LIGAND), TAP-C(O)(CH₂)_(n)NH(LIGAND), or TAP-NR″″(CH₂)_(n)NH(LIGAND); TAP-(CH₂)_(n)ONH(LIGAND), TAP-C(O)(CH₂)_(n)ONH(LIGAND), or TAP-NR″″(CH₂)_(n)ONH(LIGAND); TAP-(CH₂)_(n)NHNH₂(LIGAND), TAP-C(O)(CH₂)_(n)NHNH₂(LIGAND), or TAP-NR″″(CH₂)_(n)NHNH₂(LIGAND).

In other embodiments the tether may include an electrophilic moiety, preferably at the terminal position of the tether. Preferred electrophilic moieties include, e.g., an aldehyde, alkyl halide, mesylate, tosylate, nosylate, or brosylate, or an activated carboxylic acid ester, e.g. an NHS ester, or a pentafluorophenyl ester. Preferred tethers (underlined) include TAP-(CH₂)_(n)CHO); TAP-C(O)(CH₂)_(n)CHO; or TAP-NR″″(CH₂)_(n)CHO, in which n is 1-6 and R″″ is C₁-C₆ alkyl; or TAP-(CH₂)_(n)C(O)ONHS; TAP-C(O)(CH₂)_(n)C(O)ONHS; or TAP-NR″″(CH₂)_(n)C(O)ONHS, in which n is 1-6 and R″″ is C₁-C₆ alkyl; TAP-(CH₂)_(n)C(O)OC₆F₅ ; TAP-C(O)(CH₂)_(n)C(O)OC₆F₅ ; or TAP-NR″″(CH₂)_(n)C(O)OC₆F₅ , in which n is 1-6 and R″″ is C₁-C₆ alkyl; or —(CH₂)_(n)CH₂LG; TAP-C(O)(CH₂)_(n)CH₂LG; or TAP-NR″″(CH₂)_(n)CH₂LG, in which n is 1-6 and R″″ is C₁-C₆ alkyl (LG can be a leaving group, e.g., halide, mesylate, tosylate, nosylate, brosylate). Tethering can be carried out by coupling a nucleophilic group of a ligand, e.g., a thiol or amino group with an electrophilic group on the tether.

Tethered Entities

A wide variety of entities can be tethered to an iRNA agent, e.g., to the carrier of an RRMS. Examples are described below in the context of an RRMS but that is only preferred, entities can be coupled at other points to an iRNA agent.

Preferred moieties are ligands, which are coupled, preferably covalently, either directly or indirectly via an intervening tether, to the RRMS carrier. In preferred embodiments, the ligand is attached to the carrier via an intervening tether. As discussed above, the ligand or tethered ligand may be present on the RRMS monomer when the RRMS monomer is incorporated into the growing strand. In some embodiments, the ligand may be incorporated into a “precursor” RRMS after a “precursor” RRMS monomer has been incorporated into the growing strand. For example, an RRMS monomer having, e.g., an amino-terminated tether (i.e., having no associated ligand), e.g., TAP-(CH₂)_(n)NH₂ may be incorporated into a growing sense or antisense strand. In a subsequent operation, i.e., after incorporation of the precursor monomer into the strand, a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the precursor RRMS by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor RRMS tether.

In preferred embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g, molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.

Preferred ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified oligoribonucleotide, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; and nuclease-resistance conferring moieties. General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, bone cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g, a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

The ligand can increase the uptake of the iRNA agent into the cell by activating an inflammatory response, for example. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. Preferably, the target tissue is the liver, preferably parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a seru protein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as that or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long (see Table 1, for example).

TABLE 1 Exemplary Cell Permeation Peptides Cell Permeation Peptide Amino acid Sequence Reference Penetratin RQIKIWFQNRRMKWKK (SEQ ID NO: 6737) Derossi et al., J. Biol. Chem. 269: 10444, 1994 Tat fragment GRKKRRQRRRPPQC (SEQ ID NO: 6738) Vives et al., J. Biol. (48-60) Chem., 272: 16010, 1997 Signal GALFLGWLGAAGSTMGAWSQPKKKRKV Chaloin et al., Sequence- (SEQ ID NO: 6738) Biochem. Biophys. based peptide Res. Commun., 243: 601, 1998 PVEC LLIILRRRIRKQAHAHSK (SEQ ID NO: 6739) Elmquist et al., Exp. Cell Res., 269: 237, 2001 Transportan GWTLNSAGYLLKINLKALAALAKKIL Pooga et al., FASEB (SEQ ID NO: 6740) J., 12: 67, 1998 Amphiphilic KLALKLALKALKAALKLA (SEQ ID Oehlke et al., Mol. model peptide NO: 6741) Ther., 2: 339, 2000 Arg₉ RRRRRRRRR (SEQ ID NO: 6742) Mitchell et al., J. Pept. Res., 56: 318, 2000 Bacterial cell KFFKFFKFFK (SEQ ID NO: 6743) wall permeating LL-37 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRN LVPRTES (SEQ ID NO: 6744) Cecropin P1 SWLSKTAKKLENSAKKRISEGIAIAIQGGP R (SEQ ID NO: 6745) α-defensin ACYCRIPACIAGERRYGTCIYQGRLWAFC C (SEQ ID NO: 6746) b-defensin DHYNCVSSGGQCLYSACPIFTKIQGTCYR GKAKCCK (SEQ ID NO: 6747) Bactenecin RKCRIVVIRVCR (SEQ ID NO: 6748) PR-39 RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPP RFPPRFPGKR-NH2 (SEQ ID NO: 6749) Indolicidin ILPWKWPWWPWRR-NH2 (SEQ ID NO: 6750)

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:6751). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO:6752)) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:6753)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:6754)) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Preferably the peptide or peptidomimetic tethered to an iRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an iRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver an iRNA agent to a tumor cell expressing α_(v)β₃ (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

Peptides that target markers enriched in proliferating cells can be used. E.g., RGD containing peptides and peptidomimetics can target cancer cells, in particular cells that exhibit an I_(v)θ₃ integrin. Thus, one could use RGD peptides, cyclic peptides containing RGD, RGD peptides that include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the I_(v)θ₃ integrin ligand. Generally, such ligands can be used to control proliferating cells and angiogenesis. Preferred conjugates of this type include an iRNA agent that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancer gene described herein.

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

In one embodiment, a targeting peptide tethered to an RRMS can be an amphipathic α-helical peptide. Exemplary amphipathic α-helical peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H₂A peptides, Xenopus peptides, esculentinis-1, and caerins. A number of factors will preferably be considered to maintain the integrity of helix stability. For example, a maximum number of helix stabilization residues will be utilized (e.g., leu, ala, or lys), and a minimum number helix destabilization residues will be utilized (e.g., proline, or cyclic monomeric units. The capping residue will be considered (for example Gly is an exemplary N-capping residue and/or C-terminal amidation can be used to provide an extra H-bond to stabilize the helix. Formation of salt bridges between residues with opposite charges, separated by i±3, or i±4 positions can provide stability. For example, cationic residues such as lysine, arginine, homo-arginine, ornithine or histidine can form salt bridges with the anionic residues glutamate or aspartate.

Peptide and peptidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; α, β, or γ peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides.

Methods for Making iRNA Agents

iRNA agents can include modified or non-naturally occurring bases, e.g., bases described in copending and coowned U.S. Provisional Application Ser. No. 60/463,772, filed on Apr. 17, 2003, which is hereby incorporated by reference and/or in copending and coowned U.S. Provisional Application Ser. No. 60/465,802, filed on Apr. 25, 2003, which is hereby incorporated by reference. Monomers and iRNA agents which include such bases can be made by the methods found in U.S. Provisional Application Ser. No. 60/463,772, filed on Apr. 17, 2003, and/or in U.S. Provisional Application Ser. No. 60/465,802, filed on Apr. 25, 2003.

In addition, the invention includes iRNA agents having a modified or non-naturally occurring base and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a modified or non-naturally occurring base.

The synthesis and purification of oligonucleotide peptide conjugates can be performed by established methods. See, for example, Trufert et al., Tetrahedron, 52:3005, 1996; and Manoharan, “Oligonucleotide Conjugates in Antisense Technology,” in Antisense Drug Technology, ed. S. T. Crooke, Marcel Dekker, Inc., 2001.

In one embodiment of the invention, a peptidomimetic can be modified to create a constrained peptide that adopts a distinct and specific preferred conformation, which can increase the potency and selectivity of the peptide. For example, the constrained peptide can be an azapeptide (Gante, Synthesis, 405-413, 1989). An azapeptide is synthesized by replacing the α-carbon of an amino acid with a nitrogen atom without changing the structure of the amino acid side chain. For example, the azapeptide can be synthesized by using hydrazine in traditional peptide synthesis coupling methods, such as by reacting hydrazine with a “carbonyl donor,” e.g., phenylchloroformate.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be an N-methyl peptide. N-methyl peptides are composed of N-methyl amino acids, which provide an additional methyl group in the peptide backbone, thereby potentially providing additional means of resistance to proteolytic cleavage. N-methyl peptides can by synthesized by methods known in the art (see, for example, Lindgren et al., Trends Pharmacol. Sci. 21:99, 2000; Cell Penetrating Peptides: Processes and Applications, Langel, ed., CRC Press, Boca Raton, Fla., 2002; Fische et al., Bioconjugate. Chem. 12: 825, 2001; Wander et al., J. Am. Chem. Soc., 124:13382, 2002). For example, an Ant or Tat peptide can be an N-methyl peptide.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be a β-peptide. β-peptides form stable secondary structures such as helices, pleated sheets, turns and hairpins in solutions. Their cyclic derivatives can fold into nanotubes in the solid state. β-peptides are resistant to degradation by proteolytic enzymes. β-peptides can be synthesized by methods known in the art. For example, an Ant or Tat peptide can be a β-peptide.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be a oligocarbamate. Oligocarbamate peptides are internalized into a cell by a transport pathway facilitated by carbamate transporters. For example, an Ant or Tat peptide can be an oligocarbamate.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be an oligourea conjugate (or an oligothiourea conjugate), in which the amide bond of a peptidomimetic is replaced with a urea moiety. Replacement of the amide bond provides increased resistance to degradation by proteolytic enzymes, e.g., proteolytic enzymes in the gastrointestinal tract. In one embodiment, an oligourea conjugate is tethered to an iRNA agent for use in oral delivery. The backbone in each repeating unit of an oligourea peptidomimetic can be extended by one carbon atom in comparison with the natural amino acid. The single carbon atom extension can increase peptide stability and lipophilicity, for example. An oligourea peptide can therefore be advantageous when an iRNA agent is directed for passage through a bacterial cell wall, or when an iRNA agent must traverse the blood-brain barrier, such as for the treatment of a neurological disorder. In one embodiment, a hydrogen bonding unit is conjugated to the oligourea peptide, such as to create an increased affinity with a receptor. For example, an Ant or Tat peptide can be an oligourea conjugate (or an oligothiourea conjugate).

The siRNA peptide conjugates of the invention can be affiliated with, e.g., tethered to, RRMSs occurring at various positions on an iRNA agent. For example, a peptide can be terminally conjugated, on either the sense or the antisense strand, or a peptide can be bisconjugated (one peptide tethered to each end, one conjugated to the sense strand, and one conjugated to the antisense strand). In another option, the peptide can be internally conjugated, such as in the loop of a short hairpin iRNA agent. In yet another option, the peptide can be affiliated with a complex, such as a peptide-carrier complex.

A peptide-carrier complex consists of at least a carrier molecule, which can encapsulate one or more iRNA agents (such as for delivery to a biological system and/or a cell), and a peptide moiety tethered to the outside of the carrier molecule, such as for targeting the carrier complex to a particular tissue or cell type. A carrier complex can carry additional targeting molecules on the exterior of the complex, or fusogenic agents to aid in cell delivery. The one or more iRNA agents encapsulated within the carrier can be conjugated to lipophilic molecules, which can aid in the delivery of the agents to the interior of the carrier.

A carrier molecule or structure can be, for example, a micelle, a liposome (e.g., a cationic liposome), a nanoparticle, a microsphere, or a biodegradable polymer. A peptide moiety can be tethered to the carrier molecule by a variety of linkages, such as a disulfide linkage, an acid labile linkage, a peptide-based linkage, an oxyamino linkage or a hydrazine linkage. For example, a peptide-based linkage can be a GFLG peptide. Certain linkages will have particular advantages, and the advantages (or disadvantages) can be considered depending on the tissue target or intended use. For example, peptide based linkages are stable in the blood stream but are susceptible to enzymatic cleavage in the lysosomes.

Targeting

The iRNA agents of the invention are particularly useful when targeted to the liver. An iRNA agent can be targeted to the liver by incorporation of an RRMS containing a ligand that targets the liver. For example, a liver-targeting agent can be a lipophilic moiety. Preferred lipophilic moieties include lipid, cholesterols, oleyl, retinyl, or cholesteryl residues. Other lipophilic moieties that can function as liver-targeting agents include cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

An iRNA agent can also be targeted to the liver by association with a low-density lipoprotein (LDL), such as lactosylated LDL. Polymeric carriers complexed with sugar residues can also function to target iRNA agents to the liver.

A targeting agent that incorporates a sugar, e.g., galactose and/or analogues thereof, is particularly useful. These agents target, in particular, the parenchymal cells of the liver. For example, a targeting moiety can include more than one or preferably two or three galactose moieties, spaced about 15 angstroms from each other. The targeting moiety can alternatively be lactose (e.g., three lactose moieties), which is glucose coupled to a galactose. The targeting moiety can also be N-Acetyl-Galactosamine, N-Ac-Glucosamine. A mannose or mannose-6-phosphate targeting moiety can be used for macrophage targeting.

Conjugation of an iRNA agent with a serum albumin (SA), such as human serum albumin, can also be used to target the iRNA agent to the liver.

An iRNA agent targeted to the liver by an RRMS targeting moiety described herein can target a gene expressed in the liver. For example, the iRNA agent can target p21(WAF1/DIP1), P27(KIP1), the α-fetoprotein gene, beta-catenin, or c-MET, such as for treating a cancer of the liver. In another embodiment, the iRNA agent can target apoB-100, such as for the treatment of an HDL/LDL cholesterol imbalance; dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), or acquired hyperlipidemia; hypercholesterolemia; statin-resistant hypercholesterolemia; coronary artery disease (CAD); coronary heart disease (CHD); or atherosclerosis. In another embodiment, the iRNA agent can target forkhead homologue in rhabdomyosarcoma (FKHR); glucagon; glucagon receptor; glycogen phosphorylase; PPAR-Gamma Coactivator (PGC-1); Fructose-1,6-bisphosphatase; glucose-6-phosphatase; glucose-6-phosphate translocator; glucokinase inhibitory regulatory protein; or phosphoenolpyruvate carboxykinase (PEPCK), such as to inhibit hepatic glucose production in a mammal, such as a human, such as for the treatment of diabetes. In another embodiment, an iRNA agent targeted to the liver can target Factor V, e.g., the Leiden Factor V allele, such as to reduce the tendency to form a blood clot. An iRNA agent targeted to the liver can include a sequence which targets hepatitis virus (e.g., Hepatitis A, B, C, D, E, F, G, or H). For example, an iRNA agent of the invention can target any one of the nonstructural proteins of HCV: NS3, 4A, 4B, 5A, or 5B. For the treatment of hepatitis B, an iRNA agent can target the protein X (HBx) gene, for example.

Preferred ligands on RRMSs include folic acid, glucose, cholesterol, cholic acid, Vitamin E, Vitamin K, or Vitamin A.

DEFINITIONS

The term “halo” refers to any radical of fluorine, chlorine, bromine or iodine.

The term “alkyl” refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C₁-C₁₂ alkyl indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it. The term “haloalkyl” refers to an alkyl in which one or more hydrogen atoms are replaced by halo, and includes alkyl moieties in which all hydrogens have been replaced by halo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may be optionally inserted with O, N, or S. The terms “aralkyl” refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of “aralkyl” include benzyl, 9-fluorenyl, benzhydryl, and trityl groups.

The term “alkenyl” refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more double bonds. Examples of a typical alkenyl include, but not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. The term “alkynyl” refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more triple bonds. Some examples of a typical alkynyl are ethynyl, 2-propynyl, and 3-methylbutynyl, and propargyl. The sp² and sp³ carbons may optionally serve as the point of attachment of the alkenyl and alkynyl groups, respectively.

The term “alkoxy” refers to an —O-alkyl radical. The term “aminoalkyl” refers to an alkyl substituted with an amino. The term “mercapto” refers to an —SH radical. The term “thioalkoxy” refers to an —S-alkyl radical.

The term “alkylene” refers to a divalent alkyl (i.e., —R—), e.g., —CH₂—, —CH₂CH₂—, and —CH₂CH₂CH₂—. The term “alkylenedioxo” refers to a divalent species of the structure —O—R—O—, in which R represents an alkylene.

The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system, wherein any ring atom capable of substitution can be substituted by a substituent. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, and anthracenyl.

The term “cycloalkyl” as employed herein includes saturated cyclic, bicyclic, tricyclic, or polycyclic hydrocarbon groups having 3 to 12 carbons, wherein any ring atom capable of substitution can be substituted by a substituent. The cycloalkyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon-carbon bond. Examples of cycloalkyl moieties include, but are not limited to, cyclohexyl, adamantyl, and norbornyl.

The term “heterocyclyl” refers to a nonaromatic 3-10 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom capable of substitution can be substituted by a substituent. The heterocyclyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon-carbon bond. Examples of heterocyclyl include, but are not limited to tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino, pyrrolinyl and pyrrolidinyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom capable of substitution can be substituted by a substituent.

The term “oxo” refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted by substituents.

The term “substituents” refers to a group “substituted” on an alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group at any atom of that group. Suitable substituents include, without limitation, alkyl, alkenyl, alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, SO₃H, sulfate, phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy, ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl), S(O)_(n)alkyl (where n is 0-2), S(O)_(n) aryl (where n is 0-2), S(O)_(n) heteroaryl (where n is 0-2), S(O)_(n) heterocyclyl (where n is 0-2), amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, and combinations thereof), ester (alkyl, aralkyl, heteroaralkyl), amide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof), sulfonamide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof), unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heterocyclyl, and unsubstituted cycloalkyl. In one aspect, the substituents on a group are independently any one single, or any subset of the aforementioned substituents.

The terms “adeninyl, cytosinyl, guaninyl, thyminyl, and uracilyl” and the like refer to radicals of adenine, cytosine, guanine, thymine, and uracil.

As used herein, an “unusual” nucleobase can include any one of the following:

-   2-methyladeninyl, -   N6-methyladeninyl, -   2-methylthio-N6-methyladeninyl, -   N6-isopentenyladeninyl, -   2-methylthio-N6-isopentenyladeninyl, -   N6-(cis-hydroxyisopentenyl)adeninyl, -   2-methylthio-N6-(cis-hydroxyisopentenyl)adeninyl, -   N6-glycinylcarbamoyladeninyl, -   N6-threonylcarbamoyladeninyl, -   2-methylthio-N6-threonyl carbamoyladeninyl, -   N6-methyl-N6-threonylcarbamoyladeninyl, -   N6-hydroxynorvalylcarbamoyladeninyl, -   2-methylthio-N6-hydroxynorvalyl carbamoyladeninyl, -   N6,N6-dimethyladeninyl, -   3-methylcytosinyl, -   5-methylcytosinyl, -   2-thiocytosinyl, -   5-formylcytosinyl,

-   N4-methylcytosinyl, -   5-hydroxymethylcytosinyl, -   1-methylguaninyl, -   N2-methylguaninyl, -   7-methylguaninyl, -   N2,N2-dimethylguaninyl,

-   N2,N2,7-trimethylguaninyl, -   1-methylguaninyl, -   7-cyano-7-deazaguaninyl, -   7-aminomethyl-7-deazaguaninyl, -   pseudouracilyl, -   dihydrouracilyl, -   5-methyluracilyl, -   1-methylpseudouracilyl, -   2-thiouracilyl, -   4-thiouracilyl, -   2-thiothyminyl -   5-methyl-2-thiouracilyl, -   3-(3-amino-3-carboxypropyl)uracilyl, -   5-hydroxyuracilyl, -   5-methoxyuracilyl, -   uracilyl 5-oxyacetic acid, -   uracilyl 5-oxyacetic acid methyl ester, -   5-(carboxyhydroxymethyl)uracilyl, -   5-(carboxyhydroxymethyl)uracilyl methyl ester, -   5-methoxycarbonylmethyluracilyl, -   5-methoxycarbonylmethyl-2-thiouracilyl, -   5-aminomethyl-2-thiouracilyl, -   5-methylaminomethyluracilyl, -   5-methylaminomethyl-2-thiouracilyl, -   5-methylaminomethyl-2-selenouracilyl, -   5-carbamoylmethyluracilyl, -   5-carboxymethylaminomethyluracilyl, -   5-carboxymethylaminomethyl-2-thiouracilyl, -   3-methyluracilyl, -   1-methyl-3-(3-amino-3-carboxypropyl)pseudouracilyl, -   5-carboxymethyluracilyl, -   5-methyldihydrouracilyl, or -   3-methylpseudouracilyl.

Asymmetrical Modifications

In one aspect, the invention features an iRNA agent which can be asymmetrically modified as described herein.

In addition, the invention includes iRNA agents having asymmetrical modifications and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates an asymmetrical modification.

iRNA agents of the invention can be asymmetrically modified. An asymmetrically modified iRNA agent is one in which a strand has a modification which is not present on the other strand. An asymmetrical modification is a modification found on one strand but not on the other strand. Any modification, e.g., any modification described herein, can be present as an asymmetrical modification. An asymmetrical modification can confer any of the desired properties associated with a modification, e.g., those properties discussed herein. E.g., an asymmetrical modification can: confer resistance to degradation, an alteration in half life; target the iRNA agent to a particular target, e.g., to a particular tissue; modulate, e.g., increase or decrease, the affinity of a strand for its complement or target sequence; or hinder or promote modification of a terminal moiety, e.g., modification by a kinase or other enzymes involved in the RISC mechanism pathway. The designation of a modification as having one property does not mean that it has no other property, e.g., a modification referred to as one which promotes stabilization might also enhance targeting.

While not wishing to be bound by theory or any particular mechanistic model, it is believed that asymmetrical modification allows an iRNA agent to be optimized in view of the different or “asymmetrical” functions of the sense and antisense strands. For example, both strands can be modified to increase nuclease resistance, however, since some changes can inhibit RISC activity, these changes can be chosen for the sense stand. In addition, since some modifications, e.g., targeting moieties, can add large bulky groups that, e.g., can interfere with the cleavage activity of the RISC complex, such modifications are preferably placed on the sense strand. Thus, targeting moieties, especially bulky ones (e.g. cholesterol), are preferentially added to the sense strand. In one embodiment, an asymmetrical modification in which a phosphate of the backbone is substituted with S, e.g., a phosphorothioate modification, is present in the antisense strand, and a 2′ modification, e.g., 2′ OMe is present in the sense strand. A targeting moiety can be present at either (or both) the 5′ or 3′ end of the sense strand of the iRNA agent. In a preferred example, a P of the backbone is replaced with S in the antisense strand, 2′OMe is present in the sense strand, and a targeting moiety is added to either the 5′ or 3′ end of the sense strand of the iRNA agent.

In a preferred embodiment an asymmetrically modified iRNA agent has a modification on the sense strand which modification is not found on the antisense strand and the antisense strand has a modification which is not found on the sense strand.

Each strand can include one or more asymmetrical modifications. By way of example: one strand can include a first asymmetrical modification which confers a first property on the iRNA agent and the other strand can have a second asymmetrical modification which confers a second property on the iRNA. E.g., one strand, e.g., the sense strand can have a modification which targets the iRNA agent to a tissue, and the other strand, e.g., the antisense strand, has a modification which promotes hybridization with the target gene sequence.

In some embodiments both strands can be modified to optimize the same property, e.g., to increase resistance to nucleolytic degradation, but different modifications are chosen for the sense and the antisense strands, e.g., because the modifications affect other properties as well. E.g., since some changes can affect RISC activity these modifications are chosen for the sense strand.

In an embodiment one strand has an asymmetrical 2′ modification, e.g., a 2′ OMe modification, and the other strand has an asymmetrical modification of the phosphate backbone, e.g., a phosphorothioate modification. So, in one embodiment the antisense strand has an asymmetrical 2′ OMe modification and the sense strand has an asymmetrical phosphorothioate modification (or vice versa). In a particularly preferred embodiment the RNAi agent will have asymmetrical 2′-O alkyl, preferably, 2′-OMe modifications on the sense strand and asymmetrical backbone P modification, preferably a phosphothioate modification in the antisense strand. There can be one or multiple 2′-OMe modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunits of the sense strand can be so modified. There can be one or multiple phosphorothioate modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunits of the antisense strand can be so modified. It is preferable to have an iRNA agent wherein there are multiple 2′-OMe modifications on the sense strand and multiple phosphorothioate modifications on the antisense strand. All of the subunits on one or both strands can be so modified. A particularly preferred embodiment of multiple asymmetric modification on both strands has a duplex region about 20-21, and preferably 19, subunits in length and one or two 3′ overhangs of about 2 subunits in length.

Asymmetrical modifications are useful for promoting resistance to degradation by nucleases, e.g., endonucleases. iRNA agents can include one or more asymmetrical modifications which promote resistance to degradation. In preferred embodiments the modification on the antisense strand is one which will not interfere with silencing of the target, e.g., one which will not interfere with cleavage of the target. Most if not all sites on a strand are vulnerable, to some degree, to degradation by endonucleases. One can determine sites which are relatively vulnerable and insert asymmetrical modifications which inhibit degradation. It is often desirable to provide asymmetrical modification of a UA site in an iRNA agent, and in some cases it is desirable to provide the UA sequence on both strands with asymmetrical modification. Examples of modifications which inhibit endonucleolytic degradation can be found herein. Particularly favored modifications include: 2′ modification, e.g., provision of a 2′ OMe moiety on the U, especially on a sense strand; modification of the backbone, e.g., with the replacement of an O with an S, in the phosphate backbone, e.g., the provision of a phosphorothioate modification, on the U or the A or both, especially on an antisense strand; replacement of the U with a C5 amino linker; replacement of the A with a G (sequence changes are preferred to be located on the sense strand and not the antisense strand); and modification of the at the 2′, 6′, 7′, or 8′ position. Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications.

Asymmetrical modification can be used to inhibit degradation by exonucleases. Asymmetrical modifications can include those in which only one strand is modified as well as those in which both are modified. In preferred embodiments the modification on the antisense strand is one which will not interfere with silencing of the target, e.g., one which will not interfere with cleavage of the target. Some embodiments will have an asymmetrical modification on the sense strand, e.g., in a 3′ overhang, e.g., at the 3′ terminus, and on the antisense strand, e.g., in a 3′ overhang, e.g., at the 3′ terminus. If the modifications introduce moieties of different size it is preferable that the larger be on the sense strand. If the modifications introduce moieties of different charge it is preferable that the one with greater charge be on the sense strand.

Examples of modifications which inhibit exonucleolytic degradation can be found herein. Particularly favored modifications include: 2′ modification, e.g., provision of a 2′ OMe moiety in a 3′ overhang, e.g., at the 3′ terminus (3′ terminus means at the 3′ atom of the molecule or at the most 3′ moiety, e.g., the most 3′ P or 2′ position, as indicated by the context); modification of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P in a 3′ overhang, e.g., at the 3′ terminus; combination of a 2′ modification, e.g., provision of a 2′ O Me moiety and modification of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P, in a 3′ overhang, e.g., at the 3′ terminus; modification with a 3′ alkyl; modification with an abasic pyrolidine in a 3′ overhang, e.g., at the 3′ terminus; modification with naproxene, ibuprofen, or other moieties which inhibit degradation at the 3′ terminus. Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications.

Modifications, e.g., those described herein, which affect targeting can be provided as asymmetrical modifications. Targeting modifications which can inhibit silencing, e.g., by inhibiting cleavage of a target, can be provided as asymmetrical modifications of the sense strand. A biodistribution altering moiety, e.g., cholesterol, can be provided in one or more, e.g., two, asymmetrical modifications of the sense strand. Targeting modifications which introduce moieties having a relatively large molecular weight, e.g., a molecular weight of more than 400, 500, or 1000 daltons, or which introduce a charged moiety (e.g., having more than one positive charge or one negative charge) can be placed on the sense strand.

Modifications, e.g., those described herein, which modulate, e.g., increase or decrease, the affinity of a strand for its compliment or target, can be provided as asymmetrical modifications. These include: 5 methyl U; 5 methyl C; pseudouridine, Locked nucleic acids, 2 thio U and 2-amino-A. In some embodiments one or more of these is provided on the antisense strand.

iRNA agents have a defined structure, with a sense strand and an antisense strand, and in many cases short single strand overhangs, e.g., of 2 or 3 nucleotides are present at one or both 3′ ends. Asymmetrical modification can be used to optimize the activity of such a structure, e.g., by being placed selectively within the iRNA. E.g., the end region of the iRNA agent defined by the 5′ end of the sense strand and the 3′end of the antisense strand is important for function. This region can include the terminal 2, 3, or 4 paired nucleotides and any 3′ overhang. In preferred embodiments asymmetrical modifications which result in one or more of the following are used: modifications of the 5′ end of the sense strand which inhibit kinase activation of the sense strand, including, e.g., attachments of conjugates which target the molecule or the use modifications which protect against 5′ exonucleolytic degradation; or modifications of either strand, but preferably the sense strand, which enhance binding between the sense and antisense strand and thereby promote a “tight” structure at this end of the molecule.

The end region of the iRNA agent defined by the 3′ end of the sense strand and the 5′end of the antisense strand is also important for function. This region can include the terminal 2, 3, or 4 paired nucleotides and any 3′ overhang. Preferred embodiments include asymmetrical modifications of either strand, but preferably the sense strand, which decrease binding between the sense and antisense strand and thereby promote an “open” structure at this end of the molecule. Such modifications include placing conjugates which target the molecule or modifications which promote nuclease resistance on the sense strand in this region. Modification of the antisense strand which inhibit kinase activation are avoided in preferred embodiments.

Exemplary modifications for asymmetrical placement in the sense strand include the following:

(a) backbone modifications, e.g., modification of a backbone P, including replacement of P with S, or P substituted with alkyl or allyl, e.g., Me, and dithioates (S—P═S); these modifications can be used to promote nuclease resistance;

(b) 2′-O alkyl, e.g., 2′-OMe, 3′-O alkyl, e.g., 3′-OMe (at terminal and/or internal positions); these modifications can be used to promote nuclease resistance or to enhance binding of the sense to the antisense strand, the 3′ modifications can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;

(c) 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe and with P═O or P═S) these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;

(d) L sugars (e.g., L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe); these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;

(e) modified sugars (e.g., locked nucleic acids (LNA's), hexose nucleic acids (HNA's) and cyclohexene nucleic acids (CeNA's)); these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;

(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6 modified purines), these modifications can be used to promote nuclease resistance or to enhance binding of the sense to the antisense strand;

(g) cationic groups and Zwitterionic groups (preferably at a terminus), these modifications can be used to promote nuclease resistance;

(h) conjugate groups (preferably at terminal positions), e,g., naproxen, biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates; these modifications can be used to promote nuclease resistance or to target the molecule, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC.

Exemplary modifications for asymmetrical placement in the antisense strand include the following:

(a) backbone modifications, e.g., modification of a backbone P, including replacement of P with S, or P substituted with alkyl or allyl, e.g., Me, and dithioates (S—P═S);

(b) 2′-O alkyl, e.g., 2′-OMe, (at terminal positions);

(c) 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe) e.g., terminal at the 3′ end); e.g., with P═O or P═S preferably at the 3′-end, these modifications are preferably excluded from the 5′ end region as they may interfere with RISC enzyme activity such as kinase activity;

(d) L sugars (e.g, L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe); e.g., terminal at the 3′ end; e.g., with P═O or P═S preferably at the 3′-end, these modifications are preferably excluded from the 5′ end region as they may interfere with kinase activity;

(e) modified sugars (e.g., LNA's, HNA's and CeNA's); these modifications are preferably excluded from the 5′ end region as they may contribute to unwanted enhancements of paring between the sense and antisense strands, it is often preferred to have a “loose” structure in the 5′ region, additionally, they may interfere with kinase activity;

(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6 modified purines);

(g) cationic groups and Zwitterionic groups (preferably at a terminus);

conjugate groups (preferably at terminal positions), e,g., naproxen, biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates, but bulky groups or generally groups which inhibit RISC activity should are less preferred.

The 5′-OH of the antisense strand should be kept free to promote activity. In some preferred embodiments modifications that promote nuclease resistance should be included at the 3′ end, particularly in the 3′ overhang.

In another aspect, the invention features a method of optimizing, e.g., stabilizing, an iRNA agent. The method includes selecting a sequence having activity, introducing one or more asymmetric modifications into the sequence, wherein the introduction of the asymmetric modification optimizes a property of the iRNA agent but does not result in a decrease in activity.

The decrease in activity can be less than a preselected level of decrease. In preferred embodiments decrease in activity means a decrease of less than 5, 10, 20, 40, or 50% activity, as compared with an otherwise similar iRNA lacking the introduced modification. Activity can, e.g., be measured in vivo, or in vitro, with a result in either being sufficient to demonstrate the required maintenance of activity.

The optimized property can be any property described herein and in particular the properties discussed in the section on asymmetrical modifications provided herein. The modification can be any asymmetrical modification, e.g., an asymmetric modification described in the section on asymmetrical modifications described herein. Particularly preferred asymmetric modifications are 2′-O alkyl modifications, e.g., 2′-OMe modifications, particularly in the sense sequence, and modifications of a backbone O, particularly phosphorothioate modifications, in the antisense sequence.

In a preferred embodiment a sense sequence is selected and provided with an asymmetrical modification, while in other embodiments an antisense sequence is selected and provided with an asymmetrical modification. In some embodiments both sense and antisense sequences are selected and each provided with one or more asymmetrical modifications.

Multiple asymmetric modifications can be introduced into either or both of the sense and antisense sequence. A sequence can have at least 2, 4, 6, 8, or more modifications and all or substantially all of the monomers of a sequence can be modified.

TABLE 2 Some examples of Asymmetric Modification This table shows examples having strand I with a selected modification and strand II with a selected modification. Strand I Strand II Nuclease Resistance (e.g. 2′- Biodistribution (e.g., P = S) OMe) Biodistribution conjugate Protein Binding Functionality (e.g. Lipophile) (e.g. Naproxen) Tissue Distribution Functionality Cell Targeting Functionality (e.g. Carbohydrates) (e.g. Folate for cancer cells) Tissue Distribution Functionality Fusogenic Functionality (e.g. Liver Cell Targeting (e.g. Polyethylene imines) Carbohydrates) Cancer Cell Targeting Fusogenic Functionality (e.g. RGD peptides and imines) (e.g. peptides) Nuclease Resistance (e.g. 2′- Increase in binding Affinity (5- OMe) Me—C, 5-Me-U, 2-thio-U, 2-amino- A, G-clamp, LNA) Tissue Distribution Functionality RISC activity improving Function- ality Helical conformation changing Tissue Distribution Functionality Functionalities (P = S; lipophile, carbohydrates)

Z—X—Y Architecture

In one aspect, the invention features an iRNA agent which can have a Z—X—Y architecture or structure such as those described herein and those described in copending, co-owned U.S. Provisional Application Ser. No. 60/510,246, filed on Oct. 9, 2003, which is hereby incorporated by reference, and in copending, co-owned U.S. Provisional Application Ser. No. 60/510,318, filed on Oct. 10, 2003, which is hereby incorporated by reference.

In addition, the invention includes iRNA agents having a Z—X—Y structure and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a Z—X—Y architecture.

The invention provides an iRNA agent having a first segment, the Z region, a second segment, the X region, and optionally a third region, the Y region: Z—X—Y.

It may be desirable to modify subunits in one or both of Z and/or Y on one hand and X on the other hand. In some cases they will have the same modification or the same class of modification but it will more often be the case that the modifications made in Z and/or Y will differ from those made in X.

The Z region typically includes a terminus of an iRNA agent. The length of the Z region can vary, but will typically be from 2-14, more preferably 2-10, subunits in length. It typically is single stranded, i.e., it will not base pair with bases of another strand, though it may in some embodiments self associate, e.g., to form a loop structure. Such structures can be formed by the end of a strand looping back and forming an intrastrand duplex. E.g., 2, 3, 4, 5 or more intra-strand bases pairs can form, having a looped out or connecting region, typically of 2 or more subunits which do not pair. This can occur at one or both ends of a strand. A typical embodiment of a Z region is a single strand overhang, e.g., an over hang of the length described elsewhere herein. The Z region can thus be or include a 3′ or 5′ terminal single strand. It can be sense or antisense strand but if it is antisense it is preferred that it is a 3-overhang. Typical inter-subunit bonds in the Z region include: P═O; P═S; S—P═S; P—NR₂; and P—BR₂. Chiral P═X, where X is S, N, or B) inter-subunit bonds can also be present. (These inter-subunit bonds are discussed in more detail elsewhere herein.) Other preferred Z region subunit modifications (also discussed elsewhere herein) can include: 3′-OR, 3′SR, 2′-OMe, 3′-OMe, and 2′OH modifications and moieties; alpha configuration bases; and 2′ arabino modifications.

The X region will in most cases be duplexed, in the case of a single strand iRNA agent, with a corresponding region of the single strand, or in the case of a double stranded iRNA agent, with the corresponding region of the other strand. The length of the X region can vary but will typically be between 10-45 and more preferably between 15 and 35 subunits. Particularly preferred region X's will include 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, though other suitable lengths are described elsewhere herein and can be used. Typical X region subunits include 2′-OH subunits. In typical embodiments phosphate inter-subunit bonds are preferred while phosphorothioate or non-phosphate bonds are absent. Other modifications preferred in the X region include: modifications to improve binding, e.g., nucleobase modifications; cationic nucleobase modifications; and C-5 modified pyrimidines, e.g., allylamines. Some embodiments have 4 or more consecutive 2′OH subunits. While the use of phosphorothioate is sometimes non preferred they can be used if they connect less than 4 consecutive 2′OH subunits.

The Y region will generally conform to the parameters set out for the Z regions. However, the X and Z regions need not be the same, different types and numbers of modifications can be present, and in fact, one will usually be a 3′ overhang and one will usually be a 5′ overhang.

In a preferred embodiment the iRNA agent will have a Y and/or Z region each having ribonucleosides in which the 2′-OH is substituted, e.g., with 2′-OMe or other alkyl; and an X region that includes at least four consecutive ribonucleoside subunits in which the 2′-OH remains unsubstituted.

The subunit linkages (the linkages between subunits) of an iRNA agent can be modified, e.g., to promote resistance to degradation. Numerous examples of such modifications are disclosed herein, one example of which is the phosphorothioate linkage. These modifications can be provided between the subunits of any of the regions, Y, X, and Z. However, it is preferred that their occurrence is minimized and in particular it is preferred that consecutive modified linkages be avoided.

In a preferred embodiment the iRNA agent will have a Y and Z region each having ribonucleosides in which the 2′-OH is substituted, e.g., with 2′-OMe; and an X region that includes at least four consecutive subunits, e.g., ribonucleoside subunits in which the 2′-OH remains unsubstituted.

As mentioned above, the subunit linkages of an iRNA agent can be modified, e.g., to promote resistance to degradation. These modifications can be provided between the subunits of any of the regions, Y, X, and Z. However, it is preferred that they are minimized and in particular it is preferred that consecutive modified linkages be avoided.

Thus, in a preferred embodiment, not all of the subunit linkages of the iRNA agent are modified and more preferably the maximum number of consecutive subunits linked by other than a phosphodiester bond will be 2, 3, or 4. Particularly preferred iRNA agents will not have four or more consecutive subunits, e.g., 2′-hydroxyl ribonucleoside subunits, in which each subunits is joined by modified linkages—i.e. linkages that have been modified to stabilize them from degradation as compared to the phosphodiester linkages that naturally occur in RNA and DNA.

It is particularly preferred to minimize the occurrence in region X. Thus, in preferred embodiments each of the nucleoside subunit linkages in X will be phosphodiester linkages, or if subunit linkages in region X are modified, such modifications will be minimized. E.g., although the Y and/or Z regions can include inter subunit linkages which have been stabilized against degradation, such modifications will be minimized in the X region, and in particular consecutive modifications will be minimized. Thus, in preferred embodiments the maximum number of consecutive subunits linked by other than a phosphodiester bond will be 2, 3, or 4. Particularly preferred X regions will not have four or more consecutive subunits, e.g., 2′-hydroxyl ribonucleoside subunits, in which each subunits is joined by modified linkages—i.e. linkages that have been modified to stabilize them from degradation as compared to the phosphodiester linkages that naturally occur in RNA and DNA.

In a preferred embodiment Y and/or Z will be free of phosphorothioate linkages, though either or both may contain other modifications, e.g., other modifications of the subunit linkages.

In a preferred embodiment region X, or in some cases, the entire iRNA agent, has no more than 3 or no more than 4 subunits having identical 2′ moieties.

In a preferred embodiment region X, or in some cases, the entire iRNA agent, has no more than 3 or no more than 4 subunits having identical subunit linkages.

In a preferred embodiment one or more phosphorothioate linkages (or other modifications of the subunit linkage) are present in Y and/or Z, but such modified linkages do not connect two adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., a 2′-O-alkyl moiety. E.g., any adjacent 2′-O-alkyl moieties in the Y and/or Z, are connected by a linkage other than a phosphorothioate linkage.

In a preferred embodiment each of Y and/or Z independently has only one phosphorothioate linkage between adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides. If there is a second set of adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides, in Y and/or Z that second set is connected by a linkage other than a phosphorothioate linkage, e.g., a modified linkage other than a phosphorothioate linkage.

In a preferred embodiment each of Y and/or Z independently has more than one phosphorothioate linkage connecting adjacent pairs of subunits, e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides, but at least one pair of adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides, are be connected by a linkage other than a phosphorothioate linkage, e.g., a modified linkage other than a phosphorothioate linkage.

In a preferred embodiment one of the above recited limitation on adjacent subunits in Y and or Z is combined with a limitation on the subunits in X. E.g., one or more phosphorothioate linkages (or other modifications of the subunit linkage) are present in Y and/or Z, but such modified linkages do not connect two adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., a 2′-O-alkyl moiety. E.g., any adjacent 2′-O-alkyl moieties in the Y and/or Z, are connected by a linkage other than a phosporothioate linkage. In addition, the X region has no more than 3 or no more than 4 identical subunits, e.g., subunits having identical 2′ moieties or the X region has no more than 3 or no more than 4 subunits having identical subunit linkages.

A Y and/or Z region can include at least one, and preferably 2, 3 or 4 of a modification disclosed herein. Such modifications can be chosen, independently, from any modification described herein, e.g., from nuclease resistant subunits, subunits with modified bases, subunits with modified intersubunit linkages, subunits with modified sugars, and subunits linked to another moiety, e.g., a targeting moiety. In a preferred embodiment more than 1 of such subunits can be present but in some embodiments it is preferred that no more than 1, 2, 3, or 4 of such modifications occur, or occur consecutively. In a preferred embodiment the frequency of the modification will differ between Y and/or Z and X, e.g., the modification will be present one of Y and/or Z or X and absent in the other.

An X region can include at least one, and preferably 2, 3 or 4 of a modification disclosed herein. Such modifications can be chosen, independently, from any modification described herein, e.g., from nuclease resistant subunits, subunits with modified bases, subunits with modified intersubunit linkages, subunits with modified sugars, and subunits linked to another moiety, e.g., a targeting moiety. In a preferred embodiment more than 1 of such subunits can b present but in some embodiments it is preferred that no more than 1, 2, 3, or 4 of such modifications occur, or occur consecutively.

An RRMS (described elsewhere herein) can be introduced at one or more points in one or both strands of a double-stranded iRNA agent. An RRMS can be placed in a Y and/or Z region, at or near (within 1, 2, or 3 positions) of the 3′ or 5′ end of the sense strand or at near (within 2 or 3 positions of) the 3′ end of the antisense strand. In some embodiments it is preferred to not have an RRMS at or near (within 1, 2, or 3 positions of) the 5′ end of the antisense strand. An RRMS can be positioned in the X region, and will preferably be positioned in the sense strand or in an area of the antisense strand not critical for antisense binding to the target.

Differential Modification of Terminal Duplex Stability

In one aspect, the invention features an iRNA agent which can have differential modification of terminal duplex stability (DMTDS).

In addition, the invention includes iRNA agents having DMTDS and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates DMTDS.

iRNA agents can be optimized by increasing the propensity of the duplex to disassociate or melt (decreasing the free energy of duplex association), in the region of the 5′ end of the antisense strand duplex. This can be accomplished, e.g., by the inclusion of subunits which increase the propensity of the duplex to disassociate or melt in the region of the 5′ end of the antisense strand. It can also be accomplished by the attachment of a ligand that increases the propensity of the duplex to disassociate of melt in the region of the 5′end. While not wishing to be bound by theory, the effect may be due to promoting the effect of an enzyme such as helicase, for example, promoting the effect of the enzyme in the proximity of the 5′ end of the antisense strand.

The inventors have also discovered that iRNA agents can be optimized by decreasing the propensity of the duplex to disassociate or melt (increasing the free energy of duplex association), in the region of the 3′ end of the antisense strand duplex. This can be accomplished, e.g., by the inclusion of subunits which decrease the propensity of the duplex to disassociate or melt in the region of the 3′ end of the antisense strand. It can also be accomplished by the attachment of ligand that decreases the propensity of the duplex to disassociate of melt in the region of the 5′end.

Modifications which increase the tendency of the 5′ end of the duplex to dissociate can be used alone or in combination with other modifications described herein, e.g., with modifications which decrease the tendency of the 3′ end of the duplex to dissociate. Likewise, modifications which decrease the tendency of the 3′ end of the duplex to dissociate can be used alone or in combination with other modifications described herein, e.g., with modifications which increase the tendency of the 5′ end of the duplex to dissociate.

Decreasing the Stability of the AS 5′ End of the Duplex

Subunit pairs can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation:

-   -   A:U is preferred over G:C;     -   G:U is preferred over G:C;     -   I:C is preferred over G:C (I=inosine);     -   mismatches, e.g., non-canonical or other than canonical pairings         (as described elsewhere herein) are preferred over canonical         (A:T, A:U, G:C) pairings;     -   pairings which include a universal base are preferred over         canonical pairings.

A typical ds iRNA agent can be diagrammed as follows:

S 5′ R₁N₁N₂N₃N₄N₅ [N] N⁻⁵ N⁻⁴ N⁻³ N⁻² N⁻¹ R₂ 3′ AS 3′ R₃N₁N₂N₃N₄N₅ [N] N⁻⁵ N⁻⁴ N⁻³ N⁻² N⁻¹ R₄ 5′ S:AS P₁ P₂ P₃ P₄ P₅ [N] P⁻⁵P⁻⁴P⁻³P⁻²P⁻¹ 5′

S indicates the sense strand; AS indicates antisense strand; R₁ indicates an optional (and nonpreferred) 5′ sense strand overhang; R₂ indicates an optional (though preferred) 3′ sense overhang; R₃ indicates an optional (though preferred) 3′ antisense sense overhang; R₄ indicates an optional (and nonpreferred) 5′ antisense overhang; N indicates subunits; [N] indicates that additional subunit pairs may be present; and P_(x), indicates a paring of sense N_(x) and antisense N_(x). Overhangs are not shown in the P diagram. In some embodiments a 3′ AS overhang corresponds to region Z, the duplex region corresponds to region X, and the 3′ S strand overhang corresponds to region Y, as described elsewhere herein. (The diagram is not meant to imply maximum or minimum lengths, on which guidance is provided elsewhere herein.)

It is preferred that pairings which decrease the propensity to form a duplex are used at 1 or more of the positions in the duplex at the 5′ end of the AS strand. The terminal pair (the most 5′ pair in terms of the AS strand) is designated as P⁻¹, and the subsequent pairing positions (going in the 3′ direction in terms of the AS strand) in the duplex are designated, P⁻², P⁻³, P⁻⁴, P⁻⁵, and so on. The preferred region in which to modify to modulate duplex formation is at P⁻⁵ through P⁻¹, more preferably P⁻⁴ through P⁻¹, more preferably P⁻³ through P⁻¹. Modification at P⁻¹, is particularly preferred, alone or with modification(s) other position(s), e.g., any of the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions be chosen independently from the group of:

-   -   A:U     -   G:U     -   I:C     -   mismatched pairs, e.g., non-canonical or other than canonical         pairings or pairings which include a universal base.

In preferred embodiments the change in subunit needed to achieve a pairing which promotes dissociation will be made in the sense strand, though in some embodiments the change will be made in the antisense strand.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹, through P⁻⁴, are pairs which promote dissociation.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹, through P⁻⁴, are A:U.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹, through P⁻⁴, are G:U.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹, through P⁻⁴, are I:C.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹, through P⁻⁴, are mismatched pairs, e.g., non-canonical or other than canonical pairings.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹, through P⁻⁴, are pairings which include a universal base.

Increasing the Stability of the AS 3′ End of the Duplex

Subunit pairs can be ranked on the basis of their propensity to promote stability and inhibit dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting duplex stability:

-   -   G:C is preferred over A:U     -   Watson-Crick matches (A:T, A:U, G:C) are preferred over         non-canonical or other than canonical pairings     -   analogs that increase stability are preferred over Watson-Crick         matches (A:T, A:U, G:C)     -   2-amino-A:U is preferred over A:U     -   2-thio U or 5 Me-thio-U:A are preferred over U:A     -   G-clamp (an analog of C having 4 hydrogen bonds):G is preferred         over C:G     -   guanadinium-G-clamp:G is preferred over C:G     -   psuedo uridine:A is preferred over U:A     -   sugar modifications, e.g., 2′ modifications, e.g., 2′F, ENA, or         LNA, which enhance binding are preferred over non-modified         moieties and can be present on one or both strands to enhance         stability of the duplex. It is preferred that pairings which         increase the propensity to form a duplex are used at 1 or more         of the positions in the duplex at the 3′ end of the AS strand.         The terminal pair (the most 3′ pair in terms of the AS strand)         is designated as P₁, and the subsequent pairing positions (going         in the 5′ direction in terms of the AS strand) in the duplex are         designated, P₂, P₃, P₄, P₅, and so on. The preferred region in         which to modify to modulate duplex formation is at P₅ through         P₁, more preferably P₄ through P₁, more preferably P₃ through         P₁. Modification at P₁, is particularly preferred, alone or with         modification(s) at other position(s), e.g., any of the positions         just identified. It is preferred that at least 1, and more         preferably 2, 3, 4, or 5 of the pairs of the recited regions be         chosen independently from the group of:     -   G:C     -   a pair having an analog that increases stability over         Watson-Crick matches (A:T, A:U, G:C)     -   2-amino-A:U     -   2-thio U or 5 Me-thio-U:A     -   G-clamp (an analog of C having 4 hydrogen bonds):G     -   guanadinium-G-clamp:G     -   psuedo uridine:A     -   a pair in which one or both subunits has a sugar modification,         e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, which enhance         binding.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹, through P⁻⁴, are pairs which promote duplex stability.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁, through P₄, are G:C.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁, through P₄, are a pair having an analog that increases stability over Watson-Crick matches.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁, through P₄, are 2-amino-A:U.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁, through P₄, are 2-thio U or 5 Me-thio-U:A.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁, through P₄, are G-clamp:G.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁, through P₄, are guanidinium-G-clamp:G.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁, through P₄, are psuedo uridine:A.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁, through P₄, are a pair in which one or both subunits has a sugar modification, e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, which enhances binding.

G-clamps and guanidinium G-clamps are discussed in the following references: Holmes and Gait, “The Synthesis of 2′-O-Methyl G-Clamp Containing Oligonucleotides and Their Inhibition of the HIV-1 Tat-TAR Interaction,” Nucleosides, Nucleotides & Nucleic Acids, 22:1259-1262, 2003; Holmes et al., “Steric inhibition of human immunodeficiency virus type-1 Tat-dependent trans-activation in vitro and in cells by oligonucleotides containing 2′-O-methyl G-clamp ribonucleoside analogues,” Nucleic Acids Research, 31:2759-2768, 2003; Wilds, et al., “Structural basis for recognition of guanosine by a synthetic tricyclic cytosine analogue: Guanidinium G-clamp,” Helvetica Chimica Acta, 86:966-978, 2003; Rajeev, et al., “High-Affinity Peptide Nucleic Acid Oligomers Containing Tricyclic Cytosine Analogues,” Organic Letters, 4:4395-4398, 2002; Ausin, et al., “Synthesis of Amino- and Guanidino-G-Clamp PNA Monomers,” Organic Letters, 4:4073-4075, 2002; Maier et al., “Nuclease resistance of oligonucleotides containing the tricyclic cytosine analogues phenoxazine and 9-(2-aminoethoxy)-phenoxazine (“G-clamp”) and origins of their nuclease resistance properties,” Biochemistry, 41:1323-7, 2002; Flanagan, et al., “A cytosine analog that confers enhanced potency to antisense oligonucleotides,” Proceedings Of The National Academy Of Sciences Of The United States Of America, 96:3513-8, 1999.

Simultaneously Decreasing the Stability of the AS 5′End of the Duplex and Increasing the Stability of the AS 3′ End of the Duplex

As is discussed above, an iRNA agent can be modified to both decrease the stability of the AS 5′end of the duplex and increase the stability of the AS 3′ end of the duplex. This can be effected by combining one or more of the stability decreasing modifications in the AS 5′ end of the duplex with one or more of the stability increasing modifications in the AS 3′ end of the duplex. Accordingly a preferred embodiment includes modification in P⁻⁵ through P⁻¹, more preferably P⁻⁴ through P⁻¹ and more preferably P⁻³ through P⁻¹. Modification at P⁻¹, is particularly preferred, alone or with other position, e.g., the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions of the AS 5′ end of the duplex region be chosen independently from the group of:

A:U

G:U

I:C

mismatched pairs, e.g., non-canonical or other than canonical pairings which include a universal base; and

a modification in P₅ through P₁, more preferably P₄ through P₁ and more preferably P₃ through P₁. Modification at P₁, is particularly preferred, alone or with other position, e.g., the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions of the AS 3′ end of the duplex region be chosen independently from the group of:

G:C

a pair having an analog that increases stability over Watson-Crick matches (A:T, A:U, G:C)

2-amino-A:U

2-thio U or 5 Me-thio-U:A

G-clamp (an analog of C having 4 hydrogen bonds):G

guanadinium-G-clamp:G

psuedo uridine:A

a pair in which one or both subunits has a sugar modification, e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, which enhance binding.

The invention also includes methods of selecting and making iRNA agents having DMTDS. E.g., when screening a target sequence for candidate sequences for use as iRNA agents one can select sequences having a DMTDS property described herein or one which can be modified, preferably with as few changes as possible, especially to the

AS strand, to provide a desired level of DMTDS.

The invention also includes, providing a candidate iRNA agent sequence, and modifying at least one P in P⁻⁵ through P⁻¹ and/or at least one P in P₅ through P₁ to provide a DMTDS iRNA agent.

DMTDS iRNA agents can be used in any method described herein, e.g., to silence any gene disclosed herein, to treat any disorder described herein, in any formulation described herein, and generally in and/or with the methods and compositions described elsewhere herein. DMTDS iRNA agents can incorporate other modifications described herein, e.g., the attachment of targeting agents or the inclusion of modifications which enhance stability, e.g., the inclusion of nuclease resistant monomers or the inclusion of single strand overhangs (e.g., 3′ AS overhangs and/or 3′ S strand overhangs) which self associate to form intrastrand duplex structure.

Preferably these iRNA agents will have an architecture described herein.

Other Embodiments

In Vivo Delivery

An iRNA agent can be linked, e.g., noncovalently linked to a polymer for the efficient delivery of the iRNA agent to a subject, e.g., a mammal, such as a human. The iRNA agent can, for example, be complexed with cyclodextrin. Cyclodextrins have been used as delivery vehicles of therapeutic compounds. Cyclodextrins can form inclusion complexes with drugs that are able to fit into the hydrophobic cavity of the cyclodextrin. In other examples, cyclodextrins form non-covalent associations with other biologically active molecules such as oligonucleotides and derivatives thereof. The use of cyclodextrins creates a water-soluble drug delivery complex, that can be modified with targeting or other functional groups. Cyclodextrin cellular delivery system for oligonucleotides described in U.S. Pat. No. 5,691,316, which is hereby incorporated by reference, are suitable for use in methods of the invention. In this system, an oligonucleotide is noncovalently complexed with a cyclodextrin, or the oligonucleotide is covalently bound to adamantine which in turn is non-covalently associated with a cyclodextrin.

The delivery molecule can include a linear cyclodextrin copolymer or a linear oxidized cyclodextrin copolymer having at least one ligand bound to the cyclodextrin copolymer. Delivery systems, as described in U.S. Pat. No. 6,509,323, herein incorporated by reference, are suitable for use in methods of the invention. An iRNA agent can be bound to the linear cyclodextrin copolymer and/or a linear oxidized cyclodextrin copolymer. Either or both of the cyclodextrin or oxidized cyclodextrin copolymers can be crosslinked to another polymer and/or bound to a ligand.

A composition for iRNA delivery can employ an “inclusion complex,” a molecular compound having the characteristic structure of an adduct. In this structure, the “host molecule” spatially encloses at least part of another compound in the delivery vehicle. The enclosed compound (the “guest molecule”) is situated in the cavity of the host molecule without affecting the framework structure of the host. A “host” is preferably cyclodextrin, but can be any of the molecules suggested in U.S. Patent Publ. 2003/0008818, herein incorporated by reference.

Cyclodextrins can interact with a variety of ionic and molecular species, and the resulting inclusion compounds belong to the class of “host-guest” complexes. Within the host-guest relationship, the binding sites of the host and guest molecules should be complementary in the stereoelectronic sense. A composition of the invention can contain at least one polymer and at least one therapeutic agent, generally in the form of a particulate composite of the polymer and therapeutic agent, e.g., the iRNA agent. The iRNA agent can contain one or more complexing agents. At least one polymer of the particulate composite can interact with the complexing agent in a host-guest or a guest-host interaction to form an inclusion complex between the polymer and the complexing agent. The polymer and, more particularly, the complexing agent can be used to introduce functionality into the composition. For example, at least one polymer of the particulate composite has host functionality and forms an inclusion complex with a complexing agent having guest functionality. Alternatively, at least one polymer of the particulate composite has guest functionality and forms an inclusion complex with a complexing agent having host functionality. A polymer of the particulate composite can also contain both host and guest functionalities and form inclusion complexes with guest complexing agents and host complexing agents. A polymer with functionality can, for example, facilitate cell targeting and/or cell contact (e.g., targeting or contact to a liver cell), intercellular trafficking, and/or cell entry and release.

Upon forming the particulate composite, the iRNA agent may or may not retain its biological or therapeutic activity. Upon release from the therapeutic composition, specifically, from the polymer of the particulate composite, the activity of the iRNA agent is restored. Accordingly, the particulate composite advantageously affords the iRNA agent protection against loss of activity due to, for example, degradation and offers enhanced bioavailability. Thus, a composition may be used to provide stability, particularly storage or solution stability, to an iRNA agent or any active chemical compound. The iRNA agent may be further modified with a ligand prior to or after particulate composite or therapeutic composition formation. The ligand can provide further functionality. For example, the ligand can be a targeting moiety.

Physiological Effects

The iRNA agents described herein can be designed such that determining therapeutic toxicity is made easier by the complementarity of the iRNA agent with both a human and a non-human animal sequence. By these methods, an iRNA agent can consist of a sequence that is fully complementary to a nucleic acid sequence from a human and a nucleic acid sequence from at least one non-human animal, e.g., a non-human mammal, such as a rodent, ruminant or primate. For example, the non-human mammal can be a mouse, rat, dog, pig, goat, sheep, cow, monkey, Pan paniscus, Pan troglodytes, Macaca mulatto, or Cynomolgus monkey. The sequence of the iRNA agent could be complementary to sequences within homologous genes, e.g., oncogenes or tumor suppressor genes, of the non-human mammal and the human. By determining the toxicity of the iRNA agent in the non-human mammal, one can extrapolate the toxicity of the iRNA agent in a human. For a more strenuous toxicity test, the iRNA agent can be complementary to a human and more than one, e.g., two or three or more, non-human animals.

The methods described herein can be used to correlate any physiological effect of an iRNA agent on a human, e.g., any unwanted effect, such as a toxic effect, or any positive, or desired effect.

Delivery Module

In one aspect, the invention features a drug delivery conjugate or module, such as those described herein and those described in copending, co-owned U.S. Provisional Application Ser. No. 60/454,265, filed on Mar. 12, 2003, which is hereby incorporated by reference.

In addition, the invention includes iRNA agents described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having a chemical modification described herein, e.g., a modification which enhances resistance to degradation, an iRNA agent having an architecture or structure described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, combined with, associated with, and delivered by such a drug delivery conjugate or module.

The iRNA agents can be complexed to a delivery agent that features a modular complex. The complex can include a carrier agent linked to one or more of (preferably two or more, more preferably all three of): (a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic or electrostatic interactions); (b) a fusogenic agent (e.g., an agent capable of fusing and/or being transported through a cell membrane, e.g., an endosome membrane); and (c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell or bone cell.

An iRNA agent, e.g., iRNA agent or sRNA agent described herein, can be linked, e.g., coupled or bound, to the modular complex. The iRNA agent can interact with the condensing agent of the complex, and the complex can be used to deliver an iRNA agent to a cell, e.g., in vitro or in vivo. For example, the complex can be used to deliver an iRNA agent to a subject in need thereof, e.g., to deliver an iRNA agent to a subject having a disorder, e.g., a disorder described herein, such as a disease or disorder of the liver.

The fusogenic agent and the condensing agent can be different agents or the one and the same agent. For example, a polyamino chain, e.g., polyethyleneimine (PEI), can be the fusogenic and/or the condensing agent.

The delivery agent can be a modular complex. For example, the complex can include a carrier agent linked to one or more of (preferably two or more, more preferably all three of):

(a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic interaction),

(b) a fusogenic agent (e.g., an agent capable of fusing and/or being transported through a cell membrane, e.g., an endosome membrane), and

(c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, bone cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, Neproxin, or an RGD peptide or RGD peptide mimetic.

Carrier Agents

The carrier agent of a modular complex described herein can be a substrate for attachment of one or more of: a condensing agent, a fusogenic agent, and a targeting group. The carrier agent would preferably lack an endogenous enzymatic activity. The agent would preferably be a biological molecule, preferably a macromolecule. Polymeric biological carriers are preferred. It would also be preferred that the carrier molecule be biodegradable.

The carrier agent can be a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or lipid. The carrier molecule can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, or polyphosphazine. Other useful carrier molecules can be identified by routine methods.

A carrier agent can be characterized by one or more of: (a) is at least 1 Da in size; (b) has at least 5 charged groups, preferably between 5 and 5000 charged groups; (c) is present in the complex at a ratio of at least 1:1 carrier agent to fusogenic agent; (d) is present in the complex at a ratio of at least 1:1 carrier agent to condensing agent; (e) is present in the complex at a ratio of at least 1:1 carrier agent to targeting agent.

Fusogenic Agents

A fusogenic agent of a modular complex described herein can be an agent that is responsive to, e.g., changes charge depending on, the pH environment. Upon encountering the pH of an endosome, it can cause a physical change, e.g., a change in osmotic properties which disrupts or increases the permeability of the endosome membrane. Preferably, the fusogenic agent changes charge, e.g., becomes protonated, at pH lower than physiological range. For example, the fusogenic agent can become protonated at pH 4.5-6.5. The fusogenic agent can serve to release the iRNA agent into the cytoplasm of a cell after the complex is taken up, e.g., via endocytosis, by the cell, thereby increasing the cellular concentration of the iRNA agent in the cell.

In one embodiment, the fusogenic agent can have a moiety, e.g., an amino group, which, when exposed to a specified pH range, will undergo a change, e.g., in charge, e.g., protonation. The change in charge of the fusogenic agent can trigger a change, e.g., an osmotic change, in a vesicle, e.g., an endocytic vesicle, e.g., an endosome. For example, the fusogenic agent, upon being exposed to the pH environment of an endosome, will cause a solubility or osmotic change substantial enough to increase the porosity of (preferably, to rupture) the endosomal membrane.

The fusogenic agent can be a polymer, preferably a polyamino chain, e.g., polyethyleneimine (PEI). The PEI can be linear, branched, synthetic or natural. The PEI can be, e.g., alkyl substituted PEI, or lipid substituted PEI.

In other embodiments, the fusogenic agent can be polyhistidine, polyimidazole, polypyridine, polypropyleneimine, mellitin, or a polyacetal substance, e.g., a cationic polyacetal. In some embodiment, the fusogenic agent can have an alpha helical structure. The fusogenic agent can be a membrane disruptive agent, e.g., mellittin.

A fusogenic agent can have one or more of the following characteristics: (a) is at least 1 Da in size; (b) has at least 10 charged groups, preferably between 10 and 5000 charged groups, more preferably between 50 and 1000 charged groups; (c) is present in the complex at a ratio of at least 1:1 fusogenic agent to carrier agent; (d) is present in the complex at a ratio of at least 1:1 fusogenic agent to condensing agent; (e) is present in the complex at a ratio of at least 1:1 fusogenic agent to targeting agent.

Other suitable fusogenic agents can be tested and identified by a skilled artisan. The ability of a compound to respond to, e.g., change charge depending on, the pH environment can be tested by routine methods, e.g., in a cellular assay. For example, a test compound is combined or contacted with a cell, and the cell is allowed to take up the test compound, e.g., by endocytosis. An endosome preparation can then be made from the contacted cells and the endosome preparation compared to an endosome preparation from control cells. A change, e.g., a decrease, in the endosome fraction from the contacted cell vs. the control cell indicates that the test compound can function as a fusogenic agent. Alternatively, the contacted cell and control cell can be evaluated, e.g., by microscopy, e.g., by light or electron microscopy, to determine a difference in endosome population in the cells. The test compound can be labeled. In another type of assay, a modular complex described herein is constructed using one or more test or putative fusogenic agents. The modular complex can be constructed using a labeled nucleic acid instead of the iRNA. The ability of the fusogenic agent to respond to, e.g., change charge depending on, the pH environment, once the modular complex is taken up by the cell, can be evaluated, e.g., by preparation of an endosome preparation, or by microscopy techniques, as described above. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to respond to, e.g., change charge depending on, the pH environment; and a second assay evaluates the ability of a modular complex that includes the test compound to respond to, e.g., change charge depending on, the pH environment.

Condensing Agent

The condensing agent of a modular complex described herein can interact with (e.g., attracts, holds, or binds to) an iRNA agent and act to (a) condense, e.g., reduce the size or charge of the iRNA agent and/or (b) protect the iRNA agent, e.g., protect the iRNA agent against degradation. The condensing agent can include a moiety, e.g., a charged moiety, that can interact with a nucleic acid, e.g., an iRNA agent, e.g., by ionic interactions. The condensing agent would preferably be a charged polymer, e.g., a polycationic chain. The condensing agent can be a polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quarternary salt of a polyamine, or an alpha helical peptide.

A condensing agent can have the following characteristics: (a) at least 1 Da in size; (b) has at least 2 charged groups, preferably between 2 and 100 charged groups; (c) is present in the complex at a ratio of at least 1:1 condensing agent to carrier agent; (d) is present in the complex at a ratio of at least 1:1 condensing agent to fusogenic agent; (e) is present in the complex at a ratio of at least 1:1 condensing agent to targeting agent.

Other suitable condensing agents can be tested and identified by a skilled artisan, e.g., by evaluating the ability of a test agent to interact with a nucleic acid, e.g., an iRNA agent. The ability of a test agent to interact with a nucleic acid, e.g., an iRNA agent, e.g., to condense or protect the iRNA agent, can be evaluated by routine techniques. In one assay, a test agent is contacted with a nucleic acid, and the size and/or charge of the contacted nucleic acid is evaluated by a technique suitable to detect changes in molecular mass and/or charge. Such techniques include non-denaturing gel electrophoresis, immunological methods, e.g., immunoprecipitation, gel filtration, ionic interaction chromatography, and the like. A test agent is identified as a condensing agent if it changes the mass and/or charge (preferably both) of the contacted nucleic acid, compared to a control. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to interact with, e.g., bind to, e.g., condense the charge and/or mass of, a nucleic acid; and a second assay evaluates the ability of a modular complex that includes the test compound to interact with, e.g., bind to, e.g., condense the charge and/or mass of, a nucleic acid.

Amphipathic Delivery Agents

In one aspect, the invention features an amphipathic delivery conjugate or module, such as those described herein and those described in copending, co-owned U.S. Provisional Application Ser. No. 60/455,050, filed on Mar. 13, 2003, which is hereby incorporated by reference.

In addition, the invention include an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having a chemical modification described herein, e.g., a modification which enhances resistance to degradation, an iRNA agent having an architecture or structure described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, combined with, associated with, and delivered by such an amphipathic delivery conjugate.

An amphipathic molecule is a molecule having a hydrophobic and a hydrophilic region. Such molecules can interact with (e.g., penetrate or disrupt) lipids, e.g., a lipid bylayer of a cell. As such, they can serve as delivery agent for an associated (e.g., bound) iRNA (e.g., an iRNA or sRNA described herein). A preferred amphipathic molecule to be used in the compositions described herein (e.g., the amphipathic iRNA constructs described herein) is a polymer. The polymer may have a secondary structure, e.g., a repeating secondary structure.

One example of an amphipathic polymer is an amphipathic polypeptide, e.g., a polypeptide having a secondary structure such that the polypeptide has a hydrophilic and a hydrophobic face. The design of amphipathic peptide structures (e.g., alpha-helical polypeptides) is routine to one of skill in the art. For example, the following references provide guidance: Grell et al. (2001) Protein design and folding: template trapping of self-assembled helical bundles J Pept Sci 7(3):146-51; Chen et al. (2002) Determination of stereochemistry stability coefficients of amino acid side-chains in an amphipathic alpha-helix J Pept Res 59(1):18-33; Iwata et al. (1994) Design and synthesis of amphipathic 3(10)-helical peptides and their interactions with phospholipid bilayers and ion channel formation J Biol Chem 269(7):4928-33; Cornut et al. (1994) The amphipathic alpha-helix concept. Application to the de novo design of ideally amphipathic Leu, Lys peptides with hemolytic activity higher than that of melittin FEBS Lett 349(1):29-33; Negrete et al. (1998) Deciphering the structural code for proteins: helical propensities in domain classes and statistical multiresidue information in alpha-helices. Protein Sci 7(6):1368-79.

Another example of an amphipathic polymer is a polymer made up of two or more amphipathic subunits, e.g., two or more subunits containing cyclic moieties (e.g., a cyclic moiety having one or more hydrophilic groups and one or more hydrophobic groups). For example, the subunit may contain a steroid, e.g., cholic acid; or a aromatic moiety. Such moieties preferably can exhibit atropisomerism, such that they can form opposing hydrophobic and hydrophilic faces when in a polymer structure.

The ability of a putative amphipathic molecule to interact with a lipid membrane, e.g., a cell membrane, can be tested by routine methods, e.g., in a cell free or cellular assay. For example, a test compound is combined or contacted with a synthetic lipid bilayer, a cellular membrane fraction, or a cell, and the test compound is evaluated for its ability to interact with, penetrate or disrupt the lipid bilayer, cell membrane or cell. The test compound can labeled in order to detect the interaction with the lipid bilayer, cell membrane or cell. In another type of assay, the test compound is linked to a reporter molecule or an iRNA agent (e.g., an iRNA or sRNA described herein) and the ability of the reporter molecule or iRNA agent to penetrate the lipid bilayer, cell membrane or cell is evaluated. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to interact with a lipid bilayer, cell membrane or cell; and a second assay evaluates the ability of a construct (e.g., a construct described herein) that includes the test compound and a reporter or iRNA agent to interact with a lipid bilayer, cell membrane or cell.

An amphipathic polymer useful in the compositions described herein has at least 2, preferably at least 5, more preferably at least 10, 25, 50, 100, 200, 500, 1000, 2000, 50000 or more subunits (e.g., amino acids or cyclic subunits). A single amphipathic polymer can be linked to one or more, e.g., 2, 3, 5, 10 or more iRNA agents (e.g., iRNA or sRNA agents described herein). In some embodiments, an amphipathic polymer can contain both amino acid and cyclic subunits, e.g., aromatic subunits.

The invention features a composition that includes an iRNA agent (e.g., an iRNA or sRNA described herein) in association with an amphipathic molecule. Such compositions may be referred to herein as “amphipathic iRNA constructs.” Such compositions and constructs are useful in the delivery or targeting of iRNA agents, e.g., delivery or targeting of iRNA agents to a cell. While not wanting to be bound by theory, such compositions and constructs can increase the porosity of, e.g., can penetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g., to allow entry of the iRNA agent into a cell.

In one aspect, the invention relates to a composition comprising an iRNA agent (e.g., an iRNA or sRNA agent described herein) linked to an amphipathic molecule. The iRNA agent and the amphipathic molecule may be held in continuous contact with one another by either covalent or noncovalent linkages.

The amphipathic molecule of the composition or construct is preferably other than a phospholipid, e.g., other than a micelle, membrane or membrane fragment.

The amphipathic molecule of the composition or construct is preferably a polymer. The polymer may include two or more amphipathic subunits. One or more hydrophilic groups and one or more hydrophobic groups may be present on the polymer. The polymer may have a repeating secondary structure as well as a first face and a second face. The distribution of the hydrophilic groups and the hydrophobic groups along the repeating secondary structure can be such that one face of the polymer is a hydrophilic face and the other face of the polymer is a hydrophobic face.

The amphipathic molecule can be a polypeptide, e.g., a polypeptide comprising an α-helical conformation as its secondary structure.

In one embodiment, the amphipathic polymer includes one or more subunits containing one or more cyclic moiety (e.g., a cyclic moiety having one or more hydrophilic groups and/or one or more hydrophobic groups). In one embodiment, the polymer is a polymer of cyclic moieties such that the moieties have alternating hydrophobic and hydrophilic groups. For example, the subunit may contain a steroid, e.g., cholic acid. In another example, the subunit may contain an aromatic moiety. The aromatic moiety may be one that can exhibit atropisomerism, e.g., a 2,2′-bis(substituted)-1-1′-binaphthyl or a 2,2′-bis(substituted)biphenyl. A subunit may include an aromatic moiety of Formula (M):

The invention features a composition that includes an iRNA agent (e.g., an iRNA or sRNA described herein) in association with an amphipathic molecule. Such compositions may be referred to herein as “amphipathic iRNA constructs.” Such compositions and constructs are useful in the delivery or targeting of iRNA agents, e.g., delivery or targeting of iRNA agents to a cell. While not wanting to be bound by theory, such compositions and constructs can increase the porosity of, e.g., can penetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g., to allow entry of the iRNA agent into a cell.

In one aspect, the invention relates to a composition comprising an iRNA agent (e.g., an iRNA or sRNA agent described herein) linked to an amphipathic molecule. The iRNA agent and the amphipathic molecule may be held in continuous contact with one another by either covalent or noncovalent linkages.

The amphipathic molecule of the composition or construct is preferably other than a phospholipid, e.g., other than a micelle, membrane or membrane fragment.

The amphipathic molecule of the composition or construct is preferably a polymer. The polymer may include two or more amphipathic subunits. One or more hydrophilic groups and one or more hydrophobic groups may be present on the polymer. The polymer may have a repeating secondary structure as well as a first face and a second face. The distribution of the hydrophilic groups and the hydrophobic groups along the repeating secondary structure can be such that one face of the polymer is a hydrophilic face and the other face of the polymer is a hydrophobic face.

The amphipathic molecule can be a polypeptide, e.g., a polypeptide comprising an α-helical conformation as its secondary structure.

In one embodiment, the amphipathic polymer includes one or more subunits containing one or more cyclic moiety (e.g., a cyclic moiety having one or more hydrophilic groups and/or one or more hydrophobic groups). In one embodiment, the polymer is a polymer of cyclic moieties such that the moieties have alternating hydrophobic and hydrophilic groups. For example, the subunit may contain a steroid, e.g., cholic acid. In another example, the subunit may contain an aromatic moiety. The aromatic moiety may be one that can exhibit atropisomerism, e.g., a 2,2′-bis(substituted)-1-1′-binaphthyl or a 2,2′-bis(substituted)biphenyl. A subunit may include an aromatic moiety of Formula (M):

Referring to Formula M, R₁ is C₁-C₁₀₀ alkyl optionally substituted with aryl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl; C₁-C₁₀₀ perfluoroalkyl; or OR₅.

R₂ is hydroxy; nitro; sulfate; phosphate; phosphate ester; sulfonic acid; OR₆; or C₁-C₁₀₀ alkyl optionally substituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl or alkyl sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester, substituted or unsubstituted aryl, carboxyl, carboxylate, amino carbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S, S(O), SO₂, alkenyl, or alkynyl.

R₃ is hydrogen, or when taken together with R₄ forms a fused phenyl ring.

R₄ is hydrogen, or when taken together with R₃ forms a fused phenyl ring.

R₅ is C₁-C₁₀₀ alkyl optionally substituted with aryl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl; or C₁-C₁₀₀ perfluoroalkyl; and R₆ is C₁-C₁₀₀ alkyl optionally substituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl or alkyl sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester, substituted or unsubstituted aryl, carboxyl, carboxylate, amino carbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S, S(O), SO₂, alkenyl, or alkynyl.

Increasing Cellular Uptake of dsRNAs

A method of the invention that can include the administration of an iRNA agent and a drug that affects the uptake of the iRNA agent into the cell. The drug can be administered before, after, or at the same time that the iRNA agent is administered. The drug can be covalently linked to the iRNA agent. The drug can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB. The drug can have a transient effect on the cell.

The drug can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

The drug can also increase the uptake of the iRNA agent into the cell by activating an inflammatory response, for example. Exemplary drug's that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma interferon.

iRNA Conjugates

An iRNA agent can be coupled, e.g., covalently coupled, to a second agent. For example, an iRNA agent used to treat a particular disorder can be coupled to a second therapeutic agent, e.g., an agent other than the iRNA agent. The second therapeutic agent can be one which is directed to the treatment of the same disorder. For example, in the case of an iRNA used to treat a disorder characterized by unwanted cell proliferation, e.g., cancer, the iRNA agent can be coupled to a second agent which has an anti-cancer effect. For example, it can be coupled to an agent which stimulates the immune system, e.g., a CpG motif, or more generally an agent that activates a toll-like receptor and/or increases the production of gamma interferon.

iRNA Production

An iRNA can be produced, e.g., in bulk, by a variety of methods. Exemplary methods include: organic synthesis and RNA cleavage, e.g., in vitro cleavage.

Organic Synthesis

An iRNA can be made by separately synthesizing each respective strand of a double-stranded RNA molecule. The component strands can then be annealed.

A large bioreactor, e.g., the OligoPilot II from Pharmacia Biotec AB (Uppsala Sweden), can be used to produce a large amount of a particular RNA strand for a given iRNA. The OligoPilotII reactor can efficiently couple a nucleotide using only a 1.5 molar excess of a phosphoramidite nucleotide. To make an RNA strand, ribonucleotides amidites are used. Standard cycles of monomer addition can be used to synthesize the 21 to 23 nucleotide strand for the iRNA. Typically, the two complementary strands are produced separately and then annealed, e.g., after release from the solid support and deprotection.

Organic synthesis can be used to produce a discrete iRNA species. The complementary of the species to a particular target gene can be precisely specified. For example, the species may be complementary to a region that includes a polymorphism, e.g., a single nucleotide polymorphism. Further the location of the polymorphism can be precisely defined. In some embodiments, the polymorphism is located in an internal region, e.g., at least 4, 5, 7, or 9 nucleotides from one or both of the termini.

dsRNA Cleavage

iRNAs can also be made by cleaving a larger ds iRNA. The cleavage can be mediated in vitro or in vivo. For example, to produce iRNAs by cleavage in vitro, the following method can be used:

In vitro transcription. dsRNA is produced by transcribing a nucleic acid (DNA) segment in both directions. For example, the HiScribe™ RNAi transcription kit (New England Biolabs) provides a vector and a method for producing a dsRNA for a nucleic acid segment that is cloned into the vector at a position flanked on either side by a T7 promoter. Separate templates are generated for T7 transcription of the two complementary strands for the dsRNA. The templates are transcribed in vitro by addition of T7 RNA polymerase and dsRNA is produced. Similar methods using PCR and/or other RNA polymerases (e.g., T3 or SP6 polymerase) can also be used. In one embodiment, RNA generated by this method is carefully purified to remove endotoxins that may contaminate preparations of the recombinant enzymes.

In vitro cleavage. dsRNA is cleaved in vitro into iRNAs, for example, using a Dicer or comparable RNAse III-based activity. For example, the dsRNA can be incubated in an in vitro extract from Drosophila or using purified components, e.g. a purified RNAse or RISC complex (RNA-induced silencing complex). See, e.g., Ketting et al. Genes Dev 2001 Oct. 15; 15(20):2654-9. and Hammond Science 2001 Aug. 10; 293(5532):1146-50.

dsRNA cleavage generally produces a plurality of iRNA species, each being a particular 21 to 23 nt fragment of a source dsRNA molecule. For example, iRNAs that include sequences complementary to overlapping regions and adjacent regions of a source dsRNA molecule may be present.

Regardless of the method of synthesis, the iRNA preparation can be prepared in a solution (e.g., an aqueous and/or organic solution) that is appropriate for formulation. For example, the iRNA preparation can be precipitated and redissolved in pure double-distilled water, and lyophilized. The dried iRNA can then be resuspended in a solution appropriate for the intended formulation process.

Synthesis of modified and nucleotide surrogate iRNA agents is discussed below.

Formulation

The iRNA agents described herein can be formulated for administration to a subject

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention.

A formulated iRNA composition can assume a variety of states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the iRNA is in an aqueous phase, e.g., in a solution that includes water.

The aqueous phase or the crystalline compositions can, e.g., be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the iRNA composition is formulated in a manner that is compatible with the intended method of administration (see, below).

In particular embodiments, the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self-assembly.

A iRNA preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes a iRNA, e.g., a protein that complexes with iRNA to form an iRNP. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.

In one embodiment, the iRNA preparation includes another iRNA agent, e.g., a second iRNA that can mediated RNAi with respect to a second gene, or with respect to the same gene. Still other preparation can include at least 3, 5, ten, twenty, fifty, or a hundred or more different iRNA species. Such iRNAs can mediated RNAi with respect to a similar number of different genes.

In one embodiment, the iRNA preparation includes at least a second therapeutic agent (e.g., an agent other than an RNA or a DNA). For example, a iRNA composition for the treatment of a viral disease, e.g. HIV, might include a known antiviral agent (e.g., a protease inhibitor or reverse transcriptase inhibitor). In another example, a iRNA composition for the treatment of a cancer might further comprise a chemotherapeutic agent.

Exemplary Formulations are Discussed Below:

Liposomes

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA s agents, and such practice is within the invention. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) preparation can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the iRNA composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the iRNA are delivered into the cell where the iRNA can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the iRNA to particular cell types.

A liposome containing a iRNA can be prepared by a variety of methods.

In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNA preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the iRNA and condense around the iRNA to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of iRNA.

If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.

Further description of methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are described in, e.g., WO 96/37194. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted to packaging iRNA preparations into liposomes.

Liposomes that are pH-sensitive or negatively-charged, entrap nucleic acid molecules rather than complex with them. Since both the nucleic acid molecules and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid molecules are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 19, (1992) 269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver iRNAs to macrophages.

Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated iRNAs in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of iRNA (see, e.g., Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer iRNA, into the skin. In some implementations, liposomes are used for delivering iRNA to epidermal cells and also to enhance the penetration of iRNA into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2,405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with iRNA are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include iRNA can be delivered, for example, subcutaneously by infection in order to deliver iRNA to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading. The iRNA agents can include an RRMS tethered to a moiety which improves association with a liposome.

Surfactants

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes (see above). iRNA (or a precursor, e.g., a larger dsRNA which can be processed into a iRNA, or a DNA which encodes a iRNA or precursor) compositions can include a surfactant. In one embodiment, the iRNA is formulated as an emulsion that includes a surfactant. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

Micelles and Other Membranous Formulations

For ease of exposition the micelles and other formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these micelles and other formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. The iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)) composition can be provided as a micellar formulation. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the iRNA composition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing is preferred in order to provide smaller size micelles.

In one method a first micellar composition is prepared which contains the iRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the iRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e. there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g. through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

The preferred propellants are hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Even more preferred is HFA 134a (1,1,1,2 tetrafluoroethane).

The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g. at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

The iRNA agents can include an RRMS tethered to a moiety which improves association with a micelle or other membranous formulation.

Particles

For ease of exposition the particles, formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these particles, formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. In another embodiment, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) preparations may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques. See below for further description.

Sustained-Release Formulations. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein can be formulated for controlled, e.g., slow release. Controlled release can be achieved by disposing the iRNA within a structure or substance which impedes its release. E.g., iRNA can be disposed within a porous matrix or in an erodable matrix, either of which allow release of the iRNA over a period of time.

Polymeric particles, e.g., polymeric in microparticles can be used as a sustained-release reservoir of iRNA that is taken up by cells only released from the microparticle through biodegradation. The polymeric particles in this embodiment should therefore be large enough to preclude phagocytosis (e.g., larger than 10 μm and preferably larger than 20 μm). Such particles can be produced by the same methods to make smaller particles, but with less vigorous mixing of the first and second emulsions. That is to say, a lower homogenization speed, vortex mixing speed, or sonication setting can be used to obtain particles having a diameter around 100 μm rather than 10 μm. The time of mixing also can be altered.

Larger microparticles can be formulated as a suspension, a powder, or an implantable solid, to be delivered by intramuscular, subcutaneous, intradermal, intravenous, or intraperitoneal injection; via inhalation (intranasal or intrapulmonary); orally; or by implantation. These particles are useful for delivery of any iRNA when slow release over a relatively long term is desired. The rate of degradation, and consequently of release, varies with the polymeric formulation.

Microparticles preferably include pores, voids, hollows, defects or other interstitial spaces that allow the fluid suspension medium to freely permeate or perfuse the particulate boundary. For example, the perforated microstructures can be used to form hollow, porous spray dried microspheres.

Polymeric particles containing iRNA (e.g., a sRNA) can be made using a double emulsion technique, for instance. First, the polymer is dissolved in an organic solvent. A preferred polymer is polylactic-co-glycolic acid (PLGA), with a lactic/glycolic acid weight ratio of 65:35, 50:50, or 75:25. Next, a sample of nucleic acid suspended in aqueous solution is added to the polymer solution and the two solutions are mixed to form a first emulsion. The solutions can be mixed by vortexing or shaking, and in a preferred method, the mixture can be sonicated. Most preferable is any method by which the nucleic acid receives the least amount of damage in the form of nicking, shearing, or degradation, while still allowing the formation of an appropriate emulsion. For example, acceptable results can be obtained with a Vibra-cell model VC-250 sonicator with a ⅛″ microtip probe, at setting #3.

Spray-Drying.

An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)) can be prepared by spray drying. Spray dried iRNA can be administered to a subject or be subjected to further formulation. A pharmaceutical composition of iRNA can be prepared by spray drying a homogeneous aqueous mixture that includes a iRNA under conditions sufficient to provide a dispersible powdered composition, e.g., a pharmaceutical composition. The material for spray drying can also include one or more of: a pharmaceutically acceptable excipient, or a dispersibility-enhancing amount of a physiologically acceptable, water-soluble protein. The spray-dried product can be a dispersible powder that includes the iRNA.

Spray drying is a process that converts a liquid or slurry material to a dried particulate form. Spray drying can be used to provide powdered material for various administrative routes including inhalation. See, for example, M. Sacchetti and M. M. Van Oort in: Inhalation Aerosols: Physical and Biological Basis for Therapy, A. J. Hickey, ed. Marcel Dekkar, New York, 1996.

Spray drying can include atomizing a solution, emulsion, or suspension to form a fine mist of droplets and drying the droplets. The mist can be projected into a drying chamber (e.g., a vessel, tank, tubing, or coil) where it contacts a drying gas. The mist can include solid or liquid pore forming agents. The solvent and pore forming agents evaporate from the droplets into the drying gas to solidify the droplets, simultaneously forming pores throughout the solid. The solid (typically in a powder, particulate form) then is separated from the drying gas and collected.

Spray drying includes bringing together a highly dispersed liquid, and a sufficient volume of air (e.g., hot air) to produce evaporation and drying of the liquid droplets. The preparation to be spray dried can be any solution, course suspension, slurry, colloidal dispersion, or paste that may be atomized using the selected spray drying apparatus. Typically, the feed is sprayed into a current of warm filtered air that evaporates the solvent and conveys the dried product to a collector. The spent air is then exhausted with the solvent. Several different types of apparatus may be used to provide the desired product. For example, commercial spray dryers manufactured by Buchi Ltd. or Niro Corp. can effectively produce particles of desired size.

Spray-dried powdered particles can be approximately spherical in shape, nearly uniform in size and frequently hollow. There may be some degree of irregularity in shape depending upon the incorporated medicament and the spray drying conditions. In many instances the dispersion stability of spray-dried microspheres appears to be more effective if an inflating agent (or blowing agent) is used in their production. Particularly preferred embodiments may comprise an emulsion with an inflating agent as the disperse or continuous phase (the other phase being aqueous in nature). An inflating agent is preferably dispersed with a surfactant solution, using, for instance, a commercially available microfluidizer at a pressure of about 5000 to 15,000 psi. This process forms an emulsion, preferably stabilized by an incorporated surfactant, typically comprising submicron droplets of water immiscible blowing agent dispersed in an aqueous continuous phase. The formation of such dispersions using this and other techniques are common and well known to those in the art. The blowing agent is preferably a fluorinated compound (e.g. perfluorohexane, perfluorooctyl bromide, perfluorodecalin, perfluorobutyl ethane) which vaporizes during the spray-drying process, leaving behind generally hollow, porous aerodynamically light microspheres. As will be discussed in more detail below, other suitable blowing agents include chloroform, freons, and hydrocarbons. Nitrogen gas and carbon dioxide are also contemplated as a suitable blowing agent.

Although the perforated microstructures are preferably formed using a blowing agent as described above, it will be appreciated that, in some instances, no blowing agent is required and an aqueous dispersion of the medicament and surfactant(s) are spray dried directly. In such cases, the formulation may be amenable to process conditions (e.g., elevated temperatures) that generally lead to the formation of hollow, relatively porous microparticles. Moreover, the medicament may possess special physicochemical properties (e.g., high crystallinity, elevated melting temperature, surface activity, etc.) that make it particularly suitable for use in such techniques.

The perforated microstructures may optionally be associated with, or comprise, one or more surfactants. Moreover, miscible surfactants may optionally be combined with the suspension medium liquid phase. It will be appreciated by those skilled in the art that the use of surfactants may further increase dispersion stability, simplify formulation procedures or increase bioavailability upon administration. Of course combinations of surfactants, including the use of one or more in the liquid phase and one or more associated with the perforated microstructures are contemplated as being within the scope of the invention. By “associated with or comprise” it is meant that the structural matrix or perforated microstructure may incorporate, adsorb, absorb, be coated with or be formed by the surfactant.

Surfactants suitable for use include any compound or composition that aids in the formation and maintenance of the stabilized respiratory dispersions by forming a layer at the interface between the structural matrix and the suspension medium. The surfactant may comprise a single compound or any combination of compounds, such as in the case of co-surfactants. Particularly preferred surfactants are substantially insoluble in the propellant, nonfluorinated, and selected from the group consisting of saturated and unsaturated lipids, nonionic detergents, nonionic block copolymers, ionic surfactants, and combinations of such agents. It should be emphasized that, in addition to the aforementioned surfactants, suitable (i.e. biocompatible) fluorinated surfactants are compatible with the teachings herein and may be used to provide the desired stabilized preparations.

Lipids, including phospholipids, from both natural and synthetic sources may be used in varying concentrations to form a structural matrix. Generally, compatible lipids comprise those that have a gel to liquid crystal phase transition greater than about 40° C. Preferably, the incorporated lipids are relatively long chain (i.e. C₆-C₂₂) saturated lipids and more preferably comprise phospholipids. Exemplary phospholipids useful in the disclosed stabilized preparations comprise egg phosphatidylcholine, dilauroylphosphatidylcholine, dioleylphosphatidylcholine, dipalmitoylphosphatidyl-choline, disteroylphosphatidylcholine, short-chain phosphatidylcholines, phosphatidylethanolamine, dioleylphosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, glycolipids, ganglioside GM1, sphingomyelin, phosphatidic acid, cardiolipin; lipids bearing polymer chains such as, polyethylene glycol, chitin, hyaluronic acid, or polyvinylpyrrolidone; lipids bearing sulfonated mono-, di-, and polysaccharides; fatty acids such as palmitic acid, stearic acid, and oleic acid; cholesterol, cholesterol esters, and cholesterol hemisuccinate. Due to their excellent biocompatibility characteristics, phospholipids and combinations of phospholipids and poloxamers are particularly suitable for use in the stabilized dispersions disclosed herein.

Compatible nonionic detergents comprise: sorbitan esters including sorbitan trioleate (Spans™ 85), sorbitan sesquioleate, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene (20) sorbitan monolaurate, and polyoxyethylene (20) sorbitan monooleate, oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, glycerol esters, and sucrose esters. Other suitable nonionic detergents can be easily identified using McCutcheon's Emulsifiers and Detergents (McPublishing Co., Glen Rock, N.J.). Preferred block copolymers include diblock and triblock copolymers of polyoxyethylene and polyoxypropylene, including poloxamer 188 (Pluronic® F68), poloxamer 407 (Pluronic® F-127), and poloxamer 338. Ionic surfactants such as sodium sulfosuccinate, and fatty acid soaps may also be utilized. In preferred embodiments, the microstructures may comprise oleic acid or its alkali salt.

In addition to the aforementioned surfactants, cationic surfactants or lipids are preferred especially in the case of delivery of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). Examples of suitable cationic lipids include: DOTMA, N-[-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium-chloride; DOTAP, 1,2-dioleyloxy-3-(trimethylammonio)propane; and DOTB, 1,2-dioleyl-3-(4′-trimethylammonio)butanoyl-sn-glycerol. Polycationic amino acids such as polylysine, and polyarginine are also contemplated.

For the spraying process, such spraying methods as rotary atomization, pressure atomization and two-fluid atomization can be used. Examples of the devices used in these processes include “Parubisu [phonetic rendering] Mini-Spray GA-32” and “Parubisu Spray Drier DL-41”, manufactured by Yamato Chemical Co., or “Spray Drier CL-8,” “Spray Drier L-8,” “Spray Drier FL-12,” “Spray Drier FL-16” or “Spray Drier FL-20,” manufactured by Okawara Kakoki Co., can be used for the method of spraying using rotary-disk atomizer.

While no particular restrictions are placed on the gas used to dry the sprayed material, it is recommended to use air, nitrogen gas or an inert gas. The temperature of the inlet of the gas used to dry the sprayed materials such that it does not cause heat deactivation of the sprayed material. The range of temperatures may vary between about 50° C. to about 200° C., preferably between about 50° C. and 100° C. The temperature of the outlet gas used to dry the sprayed material, may vary between about 0° C. and about 150° C., preferably between 0° C. and 90° C., and even more preferably between 0° C. and 60° C.

The spray drying is done under conditions that result in substantially amorphous powder of homogeneous constitution having a particle size that is respirable, a low moisture content and flow characteristics that allow for ready aerosolization. Preferably the particle size of the resulting powder is such that more than about 98% of the mass is in particles having a diameter of about 10 μm or less with about 90% of the mass being in particles having a diameter less than 5 μm. Alternatively, about 95% of the mass will have particles with a diameter of less than 10 μm with about 80% of the mass of the particles having a diameter of less than 5 μm.

The dispersible pharmaceutical-based dry powders that include the iRNA preparation may optionally be combined with pharmaceutical carriers or excipients which are suitable for respiratory and pulmonary administration. Such carriers may serve simply as bulking agents when it is desired to reduce the iRNA concentration in the powder which is being delivered to a patient, but may also serve to enhance the stability of the iRNA compositions and to improve the dispersibility of the powder within a powder dispersion device in order to provide more efficient and reproducible delivery of the iRNA and to improve handling characteristics of the iRNA such as flowability and consistency to facilitate manufacturing and powder filling.

Such carrier materials may be combined with the drug prior to spray drying, i.e., by adding the carrier material to the purified bulk solution. In that way, the carrier particles will be formed simultaneously with the drug particles to produce a homogeneous powder. Alternatively, the carriers may be separately prepared in a dry powder form and combined with the dry powder drug by blending. The powder carriers will usually be crystalline (to avoid water absorption), but might in some cases be amorphous or mixtures of crystalline and amorphous. The size of the carrier particles may be selected to improve the flowability of the drug powder, typically being in the range from 25 μm to 100 μm. A preferred carrier material is crystalline lactose having a size in the above-stated range.

Powders prepared by any of the above methods will be collected from the spray dryer in a conventional manner for subsequent use. For use as pharmaceuticals and other purposes, it will frequently be desirable to disrupt any agglomerates which may have formed by screening or other conventional techniques. For pharmaceutical uses, the dry powder formulations will usually be measured into a single dose, and the single dose sealed into a package. Such packages are particularly useful for dispersion in dry powder inhalers, as described in detail below. Alternatively, the powders may be packaged in multiple-dose containers.

Methods for spray drying hydrophobic and other drugs and components are described in U.S. Pat. Nos. 5,000,888; 5,026,550; 4,670,419, 4,540,602; and 4,486,435. Bloch and Speison (1983) Pharm. Acta Helv 58:14-22 teaches spray drying of hydrochlorothiazide and chlorthalidone (lipophilic drugs) and a hydrophilic adjuvant (pentaerythritol) in azeotropic solvents of dioxane-water and 2-ethoxyethanol-water. A number of Japanese Patent application Abstracts relate to spray drying of hydrophilic-hydrophobic product combinations, including JP 806766; JP 7242568; JP 7101884; JP 7101883; JP 71018982; JP 7101881; and JP 4036233. Other foreign patent publications relevant to spray drying hydrophilic-hydrophobic product combinations include FR 2594693; DE 2209477; and WO 88/07870.

Lyophilization.

An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) preparation can be made by lyophilization. Lyophilization is a freeze-drying process in which water is sublimed from the composition after it is frozen. The particular advantage associated with the lyophilization process is that biologicals and pharmaceuticals that are relatively unstable in an aqueous solution can be dried without elevated temperatures (thereby eliminating the adverse thermal effects), and then stored in a dry state where there are few stability problems. With respect to the instant invention such techniques are particularly compatible with the incorporation of nucleic acids in perforated microstructures without compromising physiological activity. Methods for providing lyophilized particulates are known to those of skill in the art and it would clearly not require undue experimentation to provide dispersion compatible microstructures in accordance with the teachings herein. Accordingly, to the extent that lyophilization processes may be used to provide microstructures having the desired porosity and size, they are conformance with the teachings herein and are expressly contemplated as being within the scope of the instant invention.

Targeting

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNAs. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention.

In some embodiments, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) is targeted to a particular cell. For example, a liposome or particle or other structure that includes a iRNA can also include a targeting moiety that recognizes a specific molecule on a target cell. The targeting moiety can be a molecule with a specific affinity for a target cell. Targeting moieties can include antibodies directed against a protein found on the surface of a target cell, or the ligand or a receptor-binding portion of a ligand for a molecule found on the surface of a target cell. For example, the targeting moiety can recognize a cancer-specific antigen (e.g., CA15-3, CA19-9, CEA, or HER2/neu.) or a viral antigen, thus delivering the iRNA to a cancer cell or a virus-infected cell. Exemplary targeting moieties include antibodies (such as IgM, IgG, IgA, IgD, and the like, or a functional portions thereof), ligands for cell surface receptors (e.g., ectodomains thereof).

Table 3 provides a number of antigens which can be used to target selected cells.

TABLE 3 ANTIGEN Exemplary tumor tissue CEA (carcinoembryonic antigen) colon, breast, lung PSA (prostate specific antigen) prostate cancer CA-125 ovarian cancer CA 15-3 breast cancer CA 19-9 breast cancer HER2/neu breast cancer α-feto protein testicular cancer, hepatic cancer β-HCG (human chorionic testicular cancer, choriocarcinoma gonadotropin) MUC-1 breast cancer Estrogen receptor breast cancer, uterine cancer Progesterone receptor breast cancer, uterine cancer EGFr (epidermal growth factor bladder cancer receptor)

In one embodiment, the targeting moiety is attached to a liposome. For example, U.S. Pat. No. 6,245,427 describes a method for targeting a liposome using a protein or peptide. In another example, a cationic lipid component of the liposome is derivatized with a targeting moiety. For example, WO 96/37194 describes converting N-glutaryldioleoylphosphatidyl ethanolamine to a N-hydroxysuccinimide activated ester. The product was then coupled to an RGD peptide.

Genes and Diseases

In one aspect, the invention features, a method of treating a subject at risk for or afflicted with unwanted cell proliferation, e.g., malignant or nonmalignant cell proliferation. The method includes:

providing an iRNA agent, e.g., an sRNA or iRNA agent described herein, e.g., an iRNA having a structure described herein, where the iRNA is homologous to and can silence, e.g., by cleavage, a gene which promotes unwanted cell proliferation;

administering an iRNA agent, e.g., an sRNA or iRNA agent described herein to a subject, preferably a human subject,

thereby treating the subject.

In a preferred embodiment the gene is a growth factor or growth factor receptor gene, a kinase, e.g., a protein tyrosine, serine or threonine kinase gene, an adaptor protein gene, a gene encoding a G protein superfamily molecule, or a gene encoding a transcription factor.

In a preferred embodiment the iRNA agent silences the PDGF beta gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PDGF beta expression, e.g., testicular and lung cancers.

In another preferred embodiment the iRNA agent silences the Erb-B gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Erb-B expression, e.g., breast cancer.

In a preferred embodiment the iRNA agent silences the Src gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Src expression, e.g., colon cancers.

In a preferred embodiment the iRNA agent silences the CRK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted CRK expression, e.g., colon and lung cancers.

In a preferred embodiment the iRNA agent silences the GRB2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted GRB2 expression, e.g., squamous cell carcinoma.

In another preferred embodiment the iRNA agent silences the RAS gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted RAS expression, e.g., pancreatic, colon and lung cancers, and chronic leukemia.

In another preferred embodiment the iRNA agent silences the MEKK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MEKK expression, e.g., squamous cell carcinoma, melanoma or leukemia.

In another preferred embodiment the iRNA agent silences the JNK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted JNK expression, e.g., pancreatic or breast cancers.

In a preferred embodiment the iRNA agent silences the RAF gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted RAF expression, e.g., lung cancer or leukemia.

In a preferred embodiment the iRNA agent silences the Erk1/2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Erk1/2 expression, e.g., lung cancer.

In another preferred embodiment the iRNA agent silences the PCNA(p21) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PCNA expression, e.g., lung cancer.

In a preferred embodiment the iRNA agent silences the MYB gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MYB expression, e.g., colon cancer or chronic myelogenous leukemia.

In a preferred embodiment the iRNA agent silences the c-MYC gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted c-MYC expression, e.g., Burkitt's lymphoma or neuroblastoma.

In another preferred embodiment the iRNA agent silences the JUN gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted JUN expression, e.g., ovarian, prostate or breast cancers.

In another preferred embodiment the iRNA agent silences the FOS gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted FOS expression, e.g., skin or prostate cancers.

In a preferred embodiment the iRNA agent silences the BCL-2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted BCL-2 expression, e.g., lung or prostate cancers or Non-Hodgkin lymphoma.

In a preferred embodiment the iRNA agent silences the Cyclin D gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin D expression, e.g., esophageal and colon cancers.

In a preferred embodiment the iRNA agent silences the VEGF gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted VEGF expression, e.g., esophageal and colon cancers.

In a preferred embodiment the iRNA agent silences the EGFR gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted EGFR expression, e.g., breast cancer.

In another preferred embodiment the iRNA agent silences the Cyclin A gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin A expression, e.g., lung and cervical cancers.

In another preferred embodiment the iRNA agent silences the Cyclin E gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin E expression, e.g., lung and breast cancers.

In another preferred embodiment the iRNA agent silences the WNT-1 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted WNT-1 expression, e.g., basal cell carcinoma.

In another preferred embodiment the iRNA agent silences the beta-catenin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted beta-catenin expression, e.g., adenocarcinoma or hepatocellular carcinoma.

In another preferred embodiment the iRNA agent silences the c-MET gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted c-MET expression, e.g., hepatocellular carcinoma.

In another preferred embodiment the iRNA agent silences the PKC gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PKC expression, e.g., breast cancer.

In a preferred embodiment the iRNA agent silences the NFKB gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted NFKB expression, e.g., breast cancer.

In a preferred embodiment the iRNA agent silences the STAT3 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted STAT3 expression, e.g., prostate cancer.

In another preferred embodiment the iRNA agent silences the survivin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted survivin expression, e.g., cervical or pancreatic cancers.

In another preferred embodiment the iRNA agent silences the Her2/Neu gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Her2/Neu expression, e.g., breast cancer.

In another preferred embodiment the iRNA agent silences the topoisomerase I gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted topoisomerase I expression, e.g., ovarian and colon cancers.

In a preferred embodiment the iRNA agent silences the topoisomerase II alpha gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted topoisomerase II expression, e.g., breast and colon cancers.

In a preferred embodiment the iRNA agent silences mutations in the p73 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p73 expression, e.g., colorectal adenocarcinoma.

In a preferred embodiment the iRNA agent silences mutations in the p21(WAF1/CIP1) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p21(WAF1/CIP1) expression, e.g., liver cancer.

In a preferred embodiment the iRNA agent silences mutations in the p27(KIP1) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p27(KIP1) expression, e.g., liver cancer.

In a preferred embodiment the iRNA agent silences mutations in the PPM1D gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PPM1D expression, e.g., breast cancer.

In a preferred embodiment the iRNA agent silences mutations in the RAS gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted RAS expression, e.g., breast cancer.

In another preferred embodiment the iRNA agent silences mutations in the caveolin I gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted caveolin I expression, e.g., esophageal squamous cell carcinoma.

In another preferred embodiment the iRNA agent silences mutations in the MIB I gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MIB I expression, e.g., male breast carcinoma (MBC).

In another preferred embodiment the iRNA agent silences mutations in the MTAI gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MTAI expression, e.g., ovarian carcinoma.

In another preferred embodiment the iRNA agent silences mutations in the M68 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted M68 expression, e.g., human adenocarcinomas of the esophagus, stomach, colon, and rectum.

In preferred embodiments the iRNA agent silences mutations in tumor suppressor genes, and thus can be used as a method to promote apoptotic activity in combination with chemotherapeutics.

In a preferred embodiment the iRNA agent silences mutations in the p53 tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p53 expression, e.g., gall bladder, pancreatic and lung cancers.

In a preferred embodiment the iRNA agent silences mutations in the p53 family member DN-p63, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted DN-p63 expression, e.g., squamous cell carcinoma

In a preferred embodiment the iRNA agent silences mutations in the pRb tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted pRb expression, e.g., oral squamous cell carcinoma

In a preferred embodiment the iRNA agent silences mutations in the APC1 tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted APC1 expression, e.g., colon cancer.

In a preferred embodiment the iRNA agent silences mutations in the BRCA1 tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted BRCA1 expression, e.g., breast cancer.

In a preferred embodiment the iRNA agent silences mutations in the PTEN tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PTEN expression, e.g., hamartomas, gliomas, and prostate and endometrial cancers.

In a preferred embodiment the iRNA agent silences MLL fusion genes, e.g., MLL-AF9, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MLL fusion gene expression, e.g., acute leukemias.

In another preferred embodiment the iRNA agent silences the BCR/ABL fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted BCR/ABL fusion gene expression, e.g., acute and chronic leukemias.

In another preferred embodiment the iRNA agent silences the TEL/AML1 fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted TEL/AML1 fusion gene expression, e.g., childhood acute leukemia.

In another preferred embodiment the iRNA agent silences the EWS/FLI1 fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted EWS/FLI1 fusion gene expression, e.g., Ewing Sarcoma.

In another preferred embodiment the iRNA agent silences the TLS/FUS1 fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted TLS/FUS1 fusion gene expression, e.g., Myxoid liposarcoma.

In another preferred embodiment the iRNA agent silences the PAX3/FKHR fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PAX3/FKHR fusion gene expression, e.g., Myxoid liposarcoma.

In another preferred embodiment the iRNA agent silences the AML1/ETO fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted AML1/ETO fusion gene expression, e.g., acute leukemia.

In another aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with a disease or disorder that may benefit by angiogenesis inhibition e.g., cancer. The method includes:

providing an iRNA agent, e.g., an iRNA agent having a structure described herein, which iRNA agent is homologous to and can silence, e.g., by cleavage, a gene which mediates angiogenesis;

administering the iRNA agent to a subject,

thereby treating the subject.

In a preferred embodiment the iRNA agent silences the alpha v-integrin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted alpha V integrin, e.g., brain tumors or tumors of epithelial origin.

In a preferred embodiment the iRNA agent silences the Flt-1 receptor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Flt-1 receptors, eg. Cancer and rheumatoid arthritis.

In a preferred embodiment the iRNA agent silences the tubulin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted tubulin, eg. Cancer and retinal neovascularization.

In a preferred embodiment the iRNA agent silences the tubulin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted tubulin, eg. Cancer and retinal neovascularization.

In another aspect, the invention features a method of treating a subject infected with a virus or at risk for or afflicted with a disorder or disease associated with a viral infection. The method includes:

providing an iRNA agent, e.g., and iRNA agent having a structure described herein, which iRNA agent is homologous to and can silence, e.g., by cleavage, a viral gene of a cellular gene which mediates viral function, e.g., entry or growth;

administering the iRNA agent to a subject, preferably a human subject,

thereby treating the subject.

Thus, the invention provides for a method of treating patients infected by the Human Papilloma Virus (HPV) or at risk for or afflicted with a disorder mediated by HPV, e.g, cervical cancer. HPV is linked to 95% of cervical carcinomas and thus an antiviral therapy is an attractive method to treat these cancers and other symptoms of viral infection.

In a preferred embodiment, the expression of a HPV gene is reduced. In another preferred embodiment, the HPV gene is one of the group of E2, E6, or E7.

In a preferred embodiment the expression of a human gene that is required for HPV replication is reduced.

The invention also includes a method of treating patients infected by the Human Immunodeficiency Virus (HIV) or at risk for or afflicted with a disorder mediated by HIV, e.g., Acquired Immune Deficiency Syndrome (AIDS).

In a preferred embodiment, the expression of a HIV gene is reduced. In another preferred embodiment, the HIV gene is CCR5, Gag, or Rev.

In a preferred embodiment the expression of a human gene that is required for HIV replication is reduced. In another preferred embodiment, the gene is CD4 or Tsg101.

The invention also includes a method for treating patients infected by the Hepatitis B Virus (HBV) or at risk for or afflicted with a disorder mediated by HBV, e.g., cirrhosis and heptocellular carcinoma.

In a preferred embodiment, the expression of a HBV gene is reduced. In another preferred embodiment, the targeted HBV gene encodes one of the group of the tail region of the HBV core protein, the pre-cregious (pre-c) region, or the cregious (c) region. In another preferred embodiment, a targeted HBV-RNA sequence is comprised of the poly(A) tail.

In preferred embodiment the expression of a human gene that is required for HBV replication is reduced.

The invention also provides for a method of treating patients infected by the Hepatitis A Virus (HAV), or at risk for or afflicted with a disorder mediated by HAV.

In a preferred embodiment the expression of a human gene that is required for HAV replication is reduced.

The present invention provides for a method of treating patients infected by the Hepatitis C Virus (HCV), or at risk for or afflicted with a disorder mediated by HCV, e.g., cirrhosis

In a preferred embodiment, the expression of a HCV gene is reduced.

In another preferred embodiment the expression of a human gene that is required for HCV replication is reduced.

The present invention also provides for a method of treating patients infected by the any of the group of Hepatitis Viral strains comprising hepatitis D, E, F, G, or H, or patients at risk for or afflicted with a disorder mediated by any of these strains of hepatitis.

In a preferred embodiment, the expression of a Hepatitis, D, E, F, G, or H gene is reduced.

In another preferred embodiment the expression of a human gene that is required for hepatitis D, E, F, G or H replication is reduced.

Methods of the invention also provide for treating patients infected by the Respiratory Syncytial Virus (RSV) or at risk for or afflicted with a disorder mediated by RSV, e.g, lower respiratory tract infection in infants and childhood asthma, pneumonia and other complications, e.g., in the elderly.

In a preferred embodiment, the expression of a RSV gene is reduced. In another preferred embodiment, the targeted HBV gene encodes one of the group of genes N, L, or P.

In a preferred embodiment the expression of a human gene that is required for RSV replication is reduced.

Methods of the invention provide for treating patients infected by the Herpes Simplex Virus (HSV) or at risk for or afflicted with a disorder mediated by HSV, e.g, genital herpes and cold sores as well as life-threatening or sight-impairing disease mainly in immunocompromised patients.

In a preferred embodiment, the expression of a HSV gene is reduced. In another preferred embodiment, the targeted HSV gene encodes DNA polymerase or the helicase-primase.

In a preferred embodiment the expression of a human gene that is required for HSV replication is reduced.

The invention also provides a method for treating patients infected by the herpes Cytomegalovirus (CMV) or at risk for or afflicted with a disorder mediated by CMV, e.g., congenital virus infections and morbidity in immunocompromised patients.

In a preferred embodiment, the expression of a CMV gene is reduced.

In a preferred embodiment the expression of a human gene that is required for CMV replication is reduced.

Methods of the invention also provide for a method of treating patients infected by the herpes Epstein Barr Virus (EBV) or at risk for or afflicted with a disorder mediated by EBV, e.g., NK/T-cell lymphoma, non-Hodgkin lymphoma, and Hodgkin disease.

In a preferred embodiment, the expression of a EBV gene is reduced.

In a preferred embodiment the expression of a human gene that is required for EBV replication is reduced.

Methods of the invention also provide for treating patients infected by Kaposi's Sarcoma-associated Herpes Virus (KSHV), also called human herpesvirus 8, or patients at risk for or afflicted with a disorder mediated by KSHV, e.g., Kaposi's sarcoma, multicentric Castleman's disease and AIDS-associated primary effusion lymphoma.

In a preferred embodiment, the expression of a KSHV gene is reduced.

In a preferred embodiment the expression of a human gene that is required for KSHV replication is reduced.

The invention also includes a method for treating patients infected by the JC Virus (JCV) or a disease or disorder associated with this virus, e.g., progressive multifocal leukoencephalopathy (PML).

In a preferred embodiment, the expression of a JCV gene is reduced.

In preferred embodiment the expression of a human gene that is required for JCV replication is reduced.

Methods of the invention also provide for treating patients infected by the myxovirus or at risk for or afflicted with a disorder mediated by myxovirus, e.g., influenza.

In a preferred embodiment, the expression of a myxovirus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for myxovirus replication is reduced.

Methods of the invention also provide for treating patients infected by the rhinovirus or at risk for of afflicted with a disorder mediated by rhinovirus, e.g., the common cold.

In a preferred embodiment, the expression of a rhinovirus gene is reduced.

In preferred embodiment the expression of a human gene that is required for rhinovirus replication is reduced.

Methods of the invention also provide for treating patients infected by the coronavirus or at risk for of afflicted with a disorder mediated by coronavirus, e.g., the common cold.

In a preferred embodiment, the expression of a coronavirus gene is reduced.

In preferred embodiment the expression of a human gene that is required for coronavirus replication is reduced.

Methods of the invention also provide for treating patients infected by the flavivirus West Nile or at risk for or afflicted with a disorder mediated by West Nile Virus.

In a preferred embodiment, the expression of a West Nile Virus gene is reduced. In another preferred embodiment, the West Nile Virus gene is one of the group comprising E, NS3, or NS5.

In a preferred embodiment the expression of a human gene that is required for West Nile Virus replication is reduced.

Methods of the invention also provide for treating patients infected by the St. Louis Encephalitis flavivirus, or at risk for or afflicted with a disease or disorder associated with this virus, e.g., viral haemorrhagic fever or neurological disease.

In a preferred embodiment, the expression of a St. Louis Encephalitis gene is reduced.

In a preferred embodiment the expression of a human gene that is required for St. Louis Encephalitis virus replication is reduced.

Methods of the invention also provide for treating patients infected by the Tick-borne encephalitis flavivirus, or at risk for or afflicted with a disorder mediated by Tick-borne encephalitis virus, e.g., viral haemorrhagic fever and neurological disease.

In a preferred embodiment, the expression of a Tick-borne encephalitis virus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Tick-borne encephalitis virus replication is reduced.

Methods of the invention also provide for methods of treating patients infected by the Murray Valley encephalitis flavivirus, which commonly results in viral haemorrhagic fever and neurological disease.

In a preferred embodiment, the expression of a Murray Valley encephalitis virus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Murray Valley encephalitis virus replication is reduced.

The invention also includes methods for treating patients infected by the dengue flavivirus, or a disease or disorder associated with this virus, e.g., dengue haemorrhagic fever.

In a preferred embodiment, the expression of a dengue virus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for dengue virus replication is reduced.

Methods of the invention also provide for treating patients infected by the Simian Virus 40 (SV40) or at risk for or afflicted with a disorder mediated by SV40, e.g., tumorigenesis.

In a preferred embodiment, the expression of a SV40 gene is reduced.

In a preferred embodiment the expression of a human gene that is required for SV40 replication is reduced.

The invention also includes methods for treating patients infected by the Human T Cell Lymphotropic Virus (HTLV), or a disease or disorder associated with this virus, e.g., leukemia and myelopathy.

In a preferred embodiment, the expression of a HTLV gene is reduced. In another preferred embodiment the HTLV1 gene is the Tax transcriptional activator.

In a preferred embodiment the expression of a human gene that is required for HTLV replication is reduced.

Methods of the invention also provide for treating patients infected by the Moloney-Murine Leukemia Virus (Mo-MuLV) or at risk for or afflicted with a disorder mediated by Mo-MuLV, e.g., T-cell leukemia.

In a preferred embodiment, the expression of a Mo-MuLV gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Mo-MuLV replication is reduced.

Methods of the invention also provide for treating patients infected by the encephalomyocarditis virus (EMCV) or at risk for or afflicted with a disorder mediated by EMCV, e.g. myocarditis. EMCV leads to myocarditis in mice and pigs and is capable of infecting human myocardial cells. This virus is therefore a concern for patients undergoing xenotransplantation.

In a preferred embodiment, the expression of a EMCV gene is reduced.

In a preferred embodiment the expression of a human gene that is required for EMCV replication is reduced.

The invention also includes a method for treating patients infected by the measles virus (MV) or at risk for or afflicted with a disorder mediated by MV, e.g. measles.

In a preferred embodiment, the expression of a MV gene is reduced.

In a preferred embodiment the expression of a human gene that is required for MV replication is reduced.

The invention also includes a method for treating patients infected by the Vericella zoster virus (VZV) or at risk for or afflicted with a disorder mediated by VZV, e.g. chicken pox or shingles (also called zoster).

In a preferred embodiment, the expression of a VZV gene is reduced.

In a preferred embodiment the expression of a human gene that is required for VZV replication is reduced.

The invention also includes a method for treating patients infected by an adenovirus or at risk for or afflicted with a disorder mediated by an adenovirus, e.g. respiratory tract infection.

In a preferred embodiment, the expression of an adenovirus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for adenovirus replication is reduced.

The invention includes a method for treating patients infected by a yellow fever virus (YFV) or at risk for or afflicted with a disorder mediated by a YFV, e.g. respiratory tract infection.

In a preferred embodiment, the expression of a YFV gene is reduced. In another preferred embodiment, the preferred gene is one of a group that includes the E, NS2A, or NS3 genes.

In a preferred embodiment the expression of a human gene that is required for YFV replication is reduced.

Methods of the invention also provide for treating patients infected by the poliovirus or at risk for or afflicted with a disorder mediated by poliovirus, e.g., polio.

In a preferred embodiment, the expression of a poliovirus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for poliovirus replication is reduced.

Methods of the invention also provide for treating patients infected by a poxvirus or at risk for or afflicted with a disorder mediated by a poxvirus, e.g., smallpox

In a preferred embodiment, the expression of a poxvirus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for poxvirus replication is reduced.

In another, aspect the invention features methods of treating a subject infected with a pathogen, e.g., a bacterial, amoebic, parasitic, or fungal pathogen. The method includes:

providing a iRNA agent, e.g., a siRNA having a structure described herein, where siRNA is homologous to and can silence, e.g., by cleavage of a pathogen gene;

administering the iRNA agent to a subject, preferably a human subject,

thereby treating the subject.

The target gene can be one involved in growth, cell wall synthesis, protein synthesis, transcription, energy metabolism, e.g., the Krebs cycle, or toxin production.

Thus, the present invention provides for a method of treating patients infected by a plasmodium that causes malaria.

In a preferred embodiment, the expression of a plasmodium gene is reduced. In another preferred embodiment, the gene is apical membrane antigen 1 (AMA1).

In a preferred embodiment the expression of a human gene that is required for plasmodium replication is reduced.

The invention also includes methods for treating patients infected by the Mycobacterium ulcerans, or a disease or disorder associated with this pathogen, e.g. Buruli ulcers.

In a preferred embodiment, the expression of a Mycobacterium ulcerans gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Mycobacterium ulcerans replication is reduced.

The invention also includes methods for treating patients infected by the Mycobacterium tuberculosis, or a disease or disorder associated with this pathogen, e.g. tuberculosis.

In a preferred embodiment, the expression of a Mycobacterium tuberculosis gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Mycobacterium tuberculosis replication is reduced.

The invention also includes methods for treating patients infected by the Mycobacterium leprae, or a disease or disorder associated with this pathogen, e.g. leprosy.

In a preferred embodiment, the expression of a Mycobacterium leprae gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Mycobacterium leprae replication is reduced.

The invention also includes methods for treating patients infected by the bacteria Staphylococcus aureus, or a disease or disorder associated with this pathogen, e.g. infections of the skin and muscous membranes.

In a preferred embodiment, the expression of a Staphylococcus aureus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Staphylococcus aureus replication is reduced.

The invention also includes methods for treating patients infected by the bacteria Streptococcus pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection.

In a preferred embodiment, the expression of a Streptococcus pneumoniae gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Streptococcus pneumoniae replication is reduced.

The invention also includes methods for treating patients infected by the bacteria Streptococcus pyogenes, or a disease or disorder associated with this pathogen, e.g. Strep throat or Scarlet fever.

In a preferred embodiment, the expression of a Streptococcus pyogenes gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Streptococcus pyogenes replication is reduced.

The invention also includes methods for treating patients infected by the bacteria Chlamydia pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection

In a preferred embodiment, the expression of a Chlamydia pneumoniae gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Chlamydia pneumoniae replication is reduced.

The invention also includes methods for treating patients infected by the bacteria Mycoplasma pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection

In a preferred embodiment, the expression of a Mycoplasma pneumoniae gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Mycoplasma pneumoniae replication is reduced.

In one aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with a disease or disorder characterized by an unwanted immune response, e.g., an inflammatory disease or disorder, or an autoimmune disease or disorder. The method includes:

providing an iRNA agent, e.g., an iRNA agent having a structure described herein, which iRNA agent is homologous to and can silence, e.g., by cleavage, a gene which mediates an unwanted immune response;

administering the iRNA agent to a subject,

thereby treating the subject.

In a preferred embodiment the disease or disorder is an ischemia or reperfusion injury, e.g., ischemia or reperfusion injury associated with acute myocardial infarction, unstable angina, cardiopulmonary bypass, surgical intervention e.g., angioplasty, e.g., percutaneous transluminal coronary angioplasty, the response to a transplanted organ or tissue, e.g., transplanted cardiac or vascular tissue; or thrombolysis.

In a preferred embodiment the disease or disorder is restenosis, e.g., restenosis associated with surgical intervention e.g., angioplasty, e.g., percutaneous transluminal coronary angioplasty.

In a preferred embodiment the disease or disorder is Inflammatory Bowel Disease, e.g., Crohn Disease or Ulcerative Colitis.

In a preferred embodiment the disease or disorder is inflammation associated with an infection or injury.

In a preferred embodiment the disease or disorder is asthma, lupus, multiple sclerosis, diabetes, e.g., type II diabetes, arthritis, e.g., rheumatoid or psoriatic.

In particularly preferred embodiments the iRNA agent silences an integrin or co-ligand thereof, e.g., VLA4, VCAM, ICAM.

In particularly preferred embodiments the iRNA agent silences a selectin or co-ligand thereof, e.g., P-selectin, E-selectin (ELAM), I-selectin, P-selectin glycoprotein-1 (PSGL-1).

In particularly preferred embodiments the iRNA agent silences a component of the complement system, e.g., C3, C5, C3aR, C5aR, C3 convertase, C5 convertase.

In particularly preferred embodiments the iRNA agent silences a chemokine or receptor thereof, e.g., TNFI, TNFJ, IL-1I, IL-1J, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-6, IL-8, TNFRI, TNFRII, IgE, SCYA11, CCR3.

In other embodiments the iRNA agent silences GCSF, Gro1, Gro2, Gro3, PF4, MIG, Pro-Platelet Basic Protein (PPBP), MIP-1I, MIP-1J, RANTES, MCP-1, MCP-2, MCP-3, CMBKR1, CMBKR2, CMBKR3, CMBKR5, AIF-1, I-309.

In one aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with acute pain or chronic pain. The method includes:

providing an iRNA agent, which iRNA is homologous to and can silence, e.g., by cleavage, a gene which mediates the processing of pain;

administering the iRNA to a subject,

thereby treating the subject.

In particularly preferred embodiments the iRNA agent silences a component of an ion channel.

In particularly preferred embodiments the iRNA agent silences a neurotransmitter receptor or ligand.

In one aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with a neurological disease or disorder. The method includes:

providing an iRNA agent which iRNA is homologous to and can silence, e.g., by cleavage, a gene which mediates a neurological disease or disorder;

administering the to a subject,

thereby treating the subject.

In a preferred embodiment the disease or disorder is Alzheimer Disease or Parkinson Disease.

In particularly preferred embodiments the iRNA agent silences an amyloid-family gene, e.g., APP; a presenilin gene, e.g., PSEN1 and PSEN2, or I-synuclein.

In a preferred embodiment the disease or disorder is a neurodegenerative trinucleotide repeat disorder, e.g., Huntington disease, dentatorubral pallidoluysian atrophy or a spinocerebellar ataxia, e.g., SCA1, SCA2, SCA3 (Machado-Joseph disease), SCAT or SCA8.

In particularly preferred embodiments the iRNA agent silences HD, DRPLA, SCA1, SCA2, MJD1, CACNL1A4, SCA7, SCA8.

The loss of heterozygosity (LOH) can result in hemizygosity for sequence, e.g., genes, in the area of LOH. This can result in a significant genetic difference between normal and disease-state cells, e.g., cancer cells, and provides a useful difference between normal and disease-state cells, e.g., cancer cells. This difference can arise because a gene or other sequence is heterozygous in euploid cells but is hemizygous in cells having LOH. The regions of LOH will often include a gene, the loss of which promotes unwanted proliferation, e.g., a tumor suppressor gene, and other sequences including, e.g., other genes, in some cases a gene which is essential for normal function, e.g., growth. Methods of the invention rely, in part, on the specific cleavage or silencing of one allele of an essential gene with an iRNA agent of the invention. The iRNA agent is selected such that it targets the single allele of the essential gene found in the cells having LOH but does not silence the other allele, which is present in cells which do not show LOH. In essence, it discriminates between the two alleles, preferentially silencing the selected allele. In essence polymorphisms, e.g., SNPs of essential genes that are affected by LOH, are used as a target for a disorder characterized by cells having LOH, e.g., cancer cells having LOH.

E.g., one of ordinary skill in the art can identify essential genes which are in proximity to tumor suppressor genes, and which are within a LOH region which includes the tumor suppressor gene. The gene encoding the large subunit of human RNA polymerase II, POLR2A, a gene located in close proximity to the tumor suppressor gene p53, is such a gene. It frequently occurs within a region of LOH in cancer cells. Other genes that occur within LOH regions and are lost in many cancer cell types include the group comprising replication protein A 70-kDa subunit, replication protein A 32-kD, ribonucleotide reductase, thymidilate synthase, TATA associated factor 2H, ribosomal protein S14, eukaryotic initiation factor 5A, alanyl tRNA synthetase, cysteinyl tRNA synthetase, NaK ATPase, alpha-1 subunit, and transferrin receptor.

Accordingly, the invention features, a method of treating a disorder characterized by LOH, e.g., cancer. The method includes:

optionally, determining the genotype of the allele of a gene in the region of LOH and preferably determining the genotype of both alleles of the gene in a normal cell;

providing an iRNA agent which preferentially cleaves or silences the allele found in the LOH cells;

administering the iRNA to the subject,

thereby treating the disorder.

The invention also includes a iRNA agent disclosed herein, e.g, an iRNA agent which can preferentially silence, e.g., cleave, one allele of a polymorphic gene

In another aspect, the invention provides a method of cleaving or silencing more than one gene with an iRNA agent. In these embodiments the iRNA agent is selected so that it has sufficient homology to a sequence found in more than one gene. For example, the sequence AAGCTGGCCCTGGACATGGAGAT (SEQ ID NO:6736) is conserved between mouse lamin B1, lamin B2, keratin complex 2-gene 1 and lamin A/C. Thus an iRNA agent targeted to this sequence would effectively silence the entire collection of genes.

The invention also includes an iRNA agent disclosed herein, which can silence more than one gene.

Route of Delivery

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. A composition that includes a iRNA can be delivered to a subject by a variety of routes. Exemplary routes include: intravenous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular.

The iRNA molecules of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more species of iRNA and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the iRNA in aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the iRNA and mechanically introducing the DNA.

Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches. In the case of tablets, carriers that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.

Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.

For ocular administration, ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers. Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers.

Topical Delivery

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. In a preferred embodiment, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) is delivered to a subject via topical administration. “Topical administration” refers to the delivery to a subject by contacting the formulation directly to a surface of the subject. The most common form of topical delivery is to the skin, but a composition disclosed herein can also be directly applied to other surfaces of the body, e.g., to the eye, a mucous membrane, to surfaces of a body cavity or to an internal surface. As mentioned above, the most common topical delivery is to the skin. The term encompasses several routes of administration including, but not limited to, topical and transdermal. These modes of administration typically include penetration of the skin's permeability barrier and efficient delivery to the target tissue or stratum. Topical administration can be used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the composition. Topical administration can also be used as a means to selectively deliver oligonucleotides to the epidermis or dermis of a subject, or to specific strata thereof, or to an underlying tissue.

The term “skin,” as used herein, refers to the epidermis and/or dermis of an animal. Mammalian skin consists of two major, distinct layers. The outer layer of the skin is called the epidermis. The epidermis is comprised of the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale, with the stratum corneum being at the surface of the skin and the stratum basale being the deepest portion of the epidermis. The epidermis is between 50 μm and 0.2 mm thick, depending on its location on the body.

Beneath the epidermis is the dermis, which is significantly thicker than the epidermis. The dermis is primarily composed of collagen in the form of fibrous bundles. The collagenous bundles provide support for, inter alia, blood vessels, lymph capillaries, glands, nerve endings and immunologically active cells.

One of the major functions of the skin as an organ is to regulate the entry of substances into the body. The principal permeability barrier of the skin is provided by the stratum corneum, which is formed from many layers of cells in various states of differentiation. The spaces between cells in the stratum corneum is filled with different lipids arranged in lattice-like formations that provide seals to further enhance the skins permeability barrier.

The permeability barrier provided by the skin is such that it is largely impermeable to molecules having molecular weight greater than about 750 Da. For larger molecules to cross the skin's permeability barrier, mechanisms other than normal osmosis must be used.

Several factors determine the permeability of the skin to administered agents. These factors include the characteristics of the treated skin, the characteristics of the delivery agent, interactions between both the drug and delivery agent and the drug and skin, the dosage of the drug applied, the form of treatment, and the post treatment regimen. To selectively target the epidermis and dermis, it is sometimes possible to formulate a composition that comprises one or more penetration enhancers that will enable penetration of the drug to a preselected stratum.

Transdermal delivery is a valuable route for the administration of lipid soluble therapeutics. The dermis is more permeable than the epidermis and therefore absorption is much more rapid through abraded, burned or denuded skin. Inflammation and other physiologic conditions that increase blood flow to the skin also enhance transdermal adsorption. Absorption via this route may be enhanced by the use of an oily vehicle (injunction) or through the use of one or more penetration enhancers. Other effective ways to deliver a composition disclosed herein via the transdermal route include hydration of the skin and the use of controlled release topical patches. The transdermal route provides a potentially effective means to deliver a composition disclosed herein for systemic and/or local therapy.

In addition, iontophoresis (transfer of ionic solutes through biological membranes under the influence of an electric field) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 163), phonophoresis or sonophoresis (use of ultrasound to enhance the absorption of various therapeutic agents across biological membranes, notably the skin and the cornea) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 166), and optimization of vehicle characteristics relative to dose position and retention at the site of administration (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 168) may be useful methods for enhancing the transport of topically applied compositions across skin and mucosal sites.

The compositions and methods provided may also be used to examine the function of various proteins and genes in vitro in cultured or preserved dermal tissues and in animals. The invention can be thus applied to examine the function of any gene. The methods of the invention can also be used therapeutically or prophylactically. For example, for the treatment of animals that are known or suspected to suffer from diseases such as psoriasis, lichen planus, toxic epidermal necrolysis, ertythema multiforme, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, Paget's disease, Kaposi's sarcoma, pulmonary fibrosis, Lyme disease and viral, fungal and bacterial infections of the skin.

Pulmonary Delivery

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. A composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) can be administered to a subject by pulmonary delivery. Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition, preferably iRNA, within the dispersion can reach the lung where it can be readily absorbed through the alveolar region directly into blood circulation. Pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs.

Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred. One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self contained. Dry powder dispersion devices, for example, deliver drugs that may be readily formulated as dry powders. A iRNA composition may be stably stored as lyophilized or spray-dried powders by itself or in combination with suitable powder carriers. The delivery of a composition for inhalation can be mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.

The term “powder” means a composition that consists of finely dispersed solid particles that are free flowing and capable of being readily dispersed in an inhalation device and subsequently inhaled by a subject so that the particles reach the lungs to permit penetration into the alveoli. Thus, the powder is said to be “respirable.” Preferably the average particle size is less than about 10 μm in diameter preferably with a relatively uniform spheroidal shape distribution. More preferably the diameter is less than about 7.5 μm and most preferably less than about 5.0 μm. Usually the particle size distribution is between about 0.1 μm and about 5 μm in diameter, particularly about 0.3 μm to about 5 μm.

The term “dry” means that the composition has a moisture content below about 10% by weight (% w) water, usually below about 5% w and preferably less it than about 3% w. A dry composition can be such that the particles are readily dispersible in an inhalation device to form an aerosol.

The term “therapeutically effective amount” is the amount present in the composition that is needed to provide the desired level of drug in the subject to be treated to give the anticipated physiological response.

The term “physiologically effective amount” is that amount delivered to a subject to give the desired palliative or curative effect.

The term “pharmaceutically acceptable carrier” means that the carrier can be taken into the lungs with no significant adverse toxicological effects on the lungs.

The types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two.

Bulking agents that are particularly valuable include compatible carbohydrates, polypeptides, amino acids or combinations thereof. Suitable carbohydrates include monosaccharides such as galactose, D-mannose, sorbose, and the like; disaccharides, such as lactose, trehalose, and the like; cyclodextrins, such as 2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such as raffinose, maltodextrins, dextrans, and the like; alditols, such as mannitol, xylitol, and the like. A preferred group of carbohydrates includes lactose, trehalose, raffinose maltodextrins, and mannitol. Suitable polypeptides include aspartame. Amino acids include alanine and glycine, with glycine being preferred.

Additives, which are minor components of the composition of this invention, may be included for conformational stability during spray drying and for improving dispersibility of the powder. These additives include hydrophobic amino acids such as tryptophan, tyrosine, leucine, phenylalanine, and the like.

Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.

Pulmonary administration of a micellar iRNA formulation may be achieved through metered dose spray devices with propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFC and CFC propellants.

Oral or Nasal Delivery

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. Both the oral and nasal membranes offer advantages over other routes of administration. For example, drugs administered through these membranes have a rapid onset of action, provide therapeutic plasma levels, avoid first pass effect of hepatic metabolism, and avoid exposure of the drug to the hostile gastrointestinal (GI) environment. Additional advantages include easy access to the membrane sites so that the drug can be applied, localized and removed easily.

In oral delivery, compositions can be targeted to a surface of the oral cavity, e.g., to sublingual mucosa which includes the membrane of ventral surface of the tongue and the floor of the mouth or the buccal mucosa which constitutes the lining of the cheek. The sublingual mucosa is relatively permeable thus giving rapid absorption and acceptable bioavailability of many drugs. Further, the sublingual mucosa is convenient, acceptable and easily accessible.

The ability of molecules to permeate through the oral mucosa appears to be related to molecular size, lipid solubility and peptide protein ionization. Small molecules, less than 1000 daltons appear to cross mucosa rapidly. As molecular size increases, the permeability decreases rapidly. Lipid soluble compounds are more permeable than non-lipid soluble molecules. Maximum absorption occurs when molecules are un-ionized or neutral in electrical charges. Therefore charged molecules present the biggest challenges to absorption through the oral mucosae.

A pharmaceutical composition of iRNA may also be administered to the buccal cavity of a human being by spraying into the cavity, without inhalation, from a metered dose spray dispenser, a mixed micellar pharmaceutical formulation as described above and a propellant. In one embodiment, the dispenser is first shaken prior to spraying the pharmaceutical formulation and propellant into the buccal cavity.

Devices

For ease of exposition the devices, formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these devices, formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) can be disposed on or in a device, e.g., a device which implanted or otherwise placed in a subject. Exemplary devices include devices which are introduced into the vasculature, e.g., devices inserted into the lumen of a vascular tissue, or which devices themselves form a part of the vasculature, including stents, catheters, heart valves, and other vascular devices. These devices, e.g., catheters or stents, can be placed in the vasculature of the lung, heart, or leg.

Other devices include non-vascular devices, e.g., devices implanted in the peritoneum, or in organ or glandular tissue, e.g., artificial organs. The device can release a therapeutic substance in addition to a iRNA, e.g., a device can release insulin.

Other devices include artificial joints, e.g., hip joints, and other orthopedic implants.

In one embodiment, unit doses or measured doses of a composition that includes iRNA are dispensed by an implanted device. The device can include a sensor that monitors a parameter within a subject. For example, the device can include pump, e.g., and, optionally, associated electronics.

Tissue, e.g., cells or organs can be treated with An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) ex vivo and then administered or implanted in a subject.

The tissue can be autologous, allogeneic, or xenogeneic tissue. E.g., tissue can be treated to reduce graft v. host disease. In other embodiments, the tissue is allogeneic and the tissue is treated to treat a disorder characterized by unwanted gene expression in that tissue. E.g., tissue, e.g., hematopoietic cells, e.g., bone marrow hematopoietic cells, can be treated to inhibit unwanted cell proliferation.

Introduction of treated tissue, whether autologous or transplant, can be combined with other therapies.

In some implementations, the iRNA treated cells are insulated from other cells, e.g., by a semi-permeable porous barrier that prevents the cells from leaving the implant, but enables molecules from the body to reach the cells and molecules produced by the cells to enter the body. In one embodiment, the porous barrier is formed from alginate.

In one embodiment, a contraceptive device is coated with or contains an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). Exemplary devices include condoms, diaphragms, IUD (implantable uterine devices, sponges, vaginal sheaths, and birth control devices. In one embodiment, the iRNA is chosen to inactive sperm or egg. In another embodiment, the iRNA is chosen to be complementary to a viral or pathogen RNA, e.g., an RNA of an STD. In some instances, the iRNA composition can include a spermicide.

Dosage

In one aspect, the invention features a method of administering an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, to a subject (e.g., a human subject). The method includes administering a unit dose of the iRNA agent, e.g., a sRNA agent, e.g., double stranded sRNA agent that (a) the double-stranded part is 19-25 nucleotides (nt) long, preferably 21-23 nt, (b) is complementary to a target RNA (e.g., an endogenous or pathogen target RNA), and, optionally, (c) includes at least one 3′ overhang 1-5 nucleotide long. In one embodiment, the unit dose is less than 1.4 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, and less than 200 nmole of RNA agent (e.g. about 4.4×10¹⁶ copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of RNA agent per kg of bodyweight.

The defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the target RNA. The unit dose, for example, can be administered by injection (e.g., intravenous or intramuscular), an inhaled dose, or a topical application. Particularly preferred dosages are less than 2, 1, or 0.1 mg/kg of body weight.

In a preferred embodiment, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time.

In one embodiment, the effective dose is administered with other traditional therapeutic modalities. In one embodiment, the subject has a viral infection and the modality is an antiviral agent other than an iRNA agent, e.g., other than a double-stranded iRNA agent, or sRNA agent. In another embodiment, the subject has atherosclerosis and the effective dose of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, is administered in combination with, e.g., after surgical intervention, e.g., angioplasty.

In one embodiment, a subject is administered an initial dose and one or more maintenance doses of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). The maintenance dose or doses are generally lower than the initial dose, e.g., one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 μg to 1.4 mg/kg of body weight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. The maintenance doses are preferably administered no more than once every 5, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient. In preferred embodiments the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once for every 5 or 8 days. Following treatment, the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state. The dosage of the compound may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.

The effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.

In one embodiment, the iRNA agent pharmaceutical composition includes a plurality of iRNA agent species. In another embodiment, the iRNA agent species has sequences that are non-overlapping and non-adjacent to another species with respect to a naturally occurring target sequence. In another embodiment, the plurality of iRNA agent species is specific for different naturally occurring target genes. In another embodiment, the iRNA agent is allele specific.

In some cases, a patient is treated with a iRNA agent in conjunction with other therapeutic modalities. For example, a patient being treated for a viral disease, e.g. an HIV associated disease (e.g., AIDS), may be administered a iRNA agent specific for a target gene essential to the virus in conjunction with a known antiviral agent (e.g., a protease inhibitor or reverse transcriptase inhibitor). In another example, a patient being treated for cancer may be administered a iRNA agent specific for a target essential for tumor cell proliferation in conjunction with a chemotherapy.

Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compound of the invention is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight (see U.S. Pat. No. 6,107,094).

The concentration of the iRNA agent composition is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans. The concentration or amount of iRNA agent administered will depend on the parameters determined for the agent and the method of administration, e.g. nasal, buccal, pulmonary. For example, nasal formulations tend to require much lower concentrations of some ingredients in order to avoid irritation or burning of the nasal passages. It is sometimes desirable to dilute an oral formulation up to 10-100 times in order to provide a suitable nasal formulation.

Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) can include a single treatment or, preferably, can include a series of treatments. It will also be appreciated that the effective dosage of a iRNA agent such as a sRNA agent used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein. For example, the subject can be monitored after administering a iRNA agent composition. Based on information from the monitoring, an additional amount of the iRNA agent composition can be administered.

Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In some embodiments, the animal models include transgenic animals that express a human gene, e.g. a gene that produces a target RNA. The transgenic animal can be deficient for the corresponding endogenous RNA. In another embodiment, the composition for testing includes a iRNA agent that is complementary, at least in an internal region, to a sequence that is conserved between the target RNA in the animal model and the target RNA in a human.

The inventors have discovered that iRNA agents described herein can be administered to mammals, particularly large mammals such as nonhuman primates or humans in a number of ways.

In one embodiment, the administration of the iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, composition is parenteral, e.g. intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.

The invention provides methods, compositions, and kits, for rectal administration or delivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes a an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of a iRNA agent described herein, e.g., a iRNA agent having a double stranded region of less than 40, and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered rectally, e.g., introduced through the rectum into the lower or upper colon. This approach is particularly useful in the treatment of, inflammatory disorders, disorders characterized by unwanted cell proliferation, e.g., polyps, or colon cancer.

The medication can be delivered to a site in the colon by introducing a dispensing device, e.g., a flexible, camera-guided device similar to that used for inspection of the colon or removal of polyps, which includes means for delivery of the medication.

The rectal administration of the iRNA agent is by means of an enema. The iRNA agent of the enema can be dissolved in a saline or buffered solution. The rectal administration can also by means of a suppository, which can include other ingredients, e.g., an excipient, e.g., cocoa butter or hydropropylmethylcellulose.

Any of the iRNA agents described herein can be administered orally, e.g., in the form of tablets, capsules, gel capsules, lozenges, troches or liquid syrups. Further, the composition can be applied topically to a surface of the oral cavity.

Any of the iRNA agents described herein can be administered buccally. For example, the medication can be sprayed into the buccal cavity or applied directly, e.g., in a liquid, solid, or gel form to a surface in the buccal cavity. This administration is particularly desirable for the treatment of inflammations of the buccal cavity, e.g., the gums or tongue, e.g., in one embodiment, the buccal administration is by spraying into the cavity, e.g., without inhalation, from a dispenser, e.g., a metered dose spray dispenser that dispenses the pharmaceutical composition and a propellant.

Any of the iRNA agents described herein can be administered to ocular tissue. For example, the medications can be applied to the surface of the eye or nearby tissue, e.g., the inside of the eyelid. They can be applied topically, e.g., by spraying, in drops, as an eyewash, or an ointment. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. The medication can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or structure. Ocular treatment is particularly desirable for treating inflammation of the eye or nearby tissue.

Any of the iRNA agents described herein can be administered directly to the skin. For example, the medication can be applied topically or delivered in a layer of the skin, e.g., by the use of a microneedle or a battery of microneedles which penetrate into the skin, but preferably not into the underlying muscle tissue. Administration of the iRNA agent composition can be topical. Topical applications can, for example, deliver the composition to the dermis or epidermis of a subject. Topical administration can be in the form of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids or powders. A composition for topical administration can be formulated as a liposome, micelle, emulsion, or other lipophilic molecular assembly. The transdermal administration can be applied with at least one penetration enhancer, such as iontophoresis, phonophoresis, and sonophoresis.

Any of the iRNA agents described herein can be administered to the pulmonary system. Pulmonary administration can be achieved by inhalation or by the introduction of a delivery device into the pulmonary system, e.g., by introducing a delivery device which can dispense the medication. A preferred method of pulmonary delivery is by inhalation. The medication can be provided in a dispenser which delivers the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered dose of medication. The subject, or another person, can administer the medication.

Pulmonary delivery is effective not only for disorders which directly affect pulmonary tissue, but also for disorders which affect other tissue.

iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or aerosol for pulmonary delivery.

Any of the iRNA agents described herein can be administered nasally. Nasal administration can be achieved by introduction of a delivery device into the nose, e.g., by introducing a delivery device which can dispense the medication. Methods of nasal delivery include spray, aerosol, liquid, e.g., by drops, or by topical administration to a surface of the nasal cavity. The medication can be provided in a dispenser with delivery of the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered dose of medication. The subject, or another person, can administer the medication.

Nasal delivery is effective not only for disorders which directly affect nasal tissue, but also for disorders which affect other tissue

iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or for nasal delivery.

An iRNA agent can be packaged in a viral natural capsid or in a chemically or enzymatically produced artificial capsid or structure derived therefrom.

The dosage of a pharmaceutical composition including a iRNA agent can be administered in order to alleviate the symptoms of a disease state, e.g., cancer or a cardiovascular disease. A subject can be treated with the pharmaceutical composition by any of the methods mentioned above.

Gene expression in a subject can be modulated by administering a pharmaceutical composition including an iRNA agent.

A subject can be treated by administering a defined amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent) composition that is in a powdered form, e.g., a collection of microparticles, such as crystalline particles. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

A subject can be treated by administering a defined amount of an iRNA agent composition that is prepared by a method that includes spray-drying, i.e. atomizing a liquid solution, emulsion, or suspension, immediately exposing the droplets to a drying gas, and collecting the resulting porous powder particles. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

The iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), can be provided in a powdered, crystallized or other finely divided form, with or without a carrier, e.g., a micro- or nano-particle suitable for inhalation or other pulmonary delivery. This can include providing an aerosol preparation, e.g., an aerosolized spray-dried composition. The aerosol composition can be provided in and/or dispensed by a metered dose delivery device.

The subject can be treated for a condition treatable by inhalation, e.g., by aerosolizing a spray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition and inhaling the aerosolized composition. The iRNA agent can be an sRNA. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

A subject can be treated by, for example, administering a composition including an effective/defined amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), wherein the composition is prepared by a method that includes spray-drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques

In another aspect, the invention features a method that includes: evaluating a parameter related to the abundance of a transcript in a cell of a subject; comparing the evaluated parameter to a reference value; and if the evaluated parameter has a preselected relationship to the reference value (e.g., it is greater), administering a iRNA agent (or a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes a iRNA agent or precursor thereof) to the subject. In one embodiment, the iRNA agent includes a sequence that is complementary to the evaluated transcript. For example, the parameter can be a direct measure of transcript levels, a measure of a protein level, a disease or disorder symptom or characterization (e.g., rate of cell proliferation and/or tumor mass, viral load,)

In another aspect, the invention features a method that includes: administering a first amount of a composition that comprises an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) to a subject, wherein the iRNA agent includes a strand substantially complementary to a target nucleic acid; evaluating an activity associated with a protein encoded by the target nucleic acid; wherein the evaluation is used to determine if a second amount should be administered. In a preferred embodiment the method includes administering a second amount of the composition, wherein the timing of administration or dosage of the second amount is a function of the evaluating. The method can include other features described herein.

In another aspect, the invention features a method of administering a source of a double-stranded iRNA agent (ds iRNA agent) to a subject. The method includes administering or implanting a source of a ds iRNA agent, e.g., a sRNA agent, that (a) includes a double-stranded region that is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to a target RNA (e.g., an endogenous RNA or a pathogen RNA), and, optionally, (c) includes at least one 3′ overhang 1-5 nt long. In one embodiment, the source releases ds iRNA agent over time, e.g. the source is a controlled or a slow release source, e.g., a microparticle that gradually releases the ds iRNA agent. In another embodiment, the source is a pump, e.g., a pump that includes a sensor or a pump that can release one or more unit doses.

In one aspect, the invention features a pharmaceutical composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) including a nucleotide sequence complementary to a target RNA, e.g., substantially and/or exactly complementary. The target RNA can be a transcript of an endogenous human gene. In one embodiment, the iRNA agent (a) is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and, optionally, (c) includes at least one 3′ overhang 1-5 nt long. In one embodiment, the pharmaceutical composition can be an emulsion, microemulsion, cream, jelly, or liposome.

In one example the pharmaceutical composition includes an iRNA agent mixed with a topical delivery agent. The topical delivery agent can be a plurality of microscopic vesicles. The microscopic vesicles can be liposomes. In a preferred embodiment the liposomes are cationic liposomes.

In another aspect, the pharmaceutical composition includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) admixed with a topical penetration enhancer. In one embodiment, the topical penetration enhancer is a fatty acid. The fatty acid can be arachidonic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₁₀ alkyl ester, monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.

In another embodiment, the topical penetration enhancer is a bile salt. The bile salt can be cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate, polyoxyethylene-9-lauryl ether or a pharmaceutically acceptable salt thereof.

In another embodiment, the penetration enhancer is a chelating agent. The chelating agent can be EDTA, citric acid, a salicylate, a N-acyl derivative of collagen, laureth-9, an N-amino acyl derivative of a beta-diketone or a mixture thereof.

In another embodiment, the penetration enhancer is a surfactant, e.g., an ionic or nonionic surfactant. The surfactant can be sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, a perfluorochemical emulsion or mixture thereof.

In another embodiment, the penetration enhancer can be selected from a group consisting of unsaturated cyclic ureas, 1-alkyl-alkones, 1-alkenylazacyclo-alakanones, steroidal anti-inflammatory agents and mixtures thereof. In yet another embodiment the penetration enhancer can be a glycol, a pyrrol, an azone, or a terpenes.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a form suitable for oral delivery. In one embodiment, oral delivery can be used to deliver an iRNA agent composition to a cell or a region of the gastro-intestinal tract, e.g., small intestine, colon (e.g., to treat a colon cancer), and so forth. The oral delivery form can be tablets, capsules or gel capsules. In one embodiment, the iRNA agent of the pharmaceutical composition modulates expression of a cellular adhesion protein, modulates a rate of cellular proliferation, or has biological activity against eukaryotic pathogens or retroviruses. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In a preferred embodiment the enteric material is a coating. The coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methylcellulose phthalate or cellulose acetate phthalate.

In another embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer. The penetration enhancer can be a bile salt or a fatty acid. The bile salt can be ursodeoxycholic acid, chenodeoxycholic acid, and salts thereof. The fatty acid can be capric acid, lauric acid, and salts thereof.

In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example the excipient is polyethyleneglycol. In another example the excipient is precirol.

In another embodiment, the oral dosage form of the pharmaceutical composition includes a plasticizer. The plasticizer can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethyl citrate.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent and a delivery vehicle. In one embodiment, the iRNA agent is (a) is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and, optionally, (c) includes at least one 3′ overhang 1-5 nucleotides long.

In one embodiment, the delivery vehicle can deliver an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) to a cell by a topical route of administration. The delivery vehicle can be microscopic vesicles. In one example the microscopic vesicles are liposomes. In a preferred embodiment the liposomes are cationic liposomes. In another example the microscopic vesicles are micelles. In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in an injectable dosage form. In one embodiment, the injectable dosage form of the pharmaceutical composition includes sterile aqueous solutions or dispersions and sterile powders. In a preferred embodiment the sterile solution can include a diluent such as water; saline solution; fixed oils, polyethylene glycols, glycerin, or propylene glycol.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in oral dosage form. In one embodiment, the oral dosage form is selected from the group consisting of tablets, capsules and gel capsules. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In a preferred embodiment the enteric material is a coating. The coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methyl cellulose phthalate or cellulose acetate phthalate. In one embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer, e.g., a penetration enhancer described herein.

In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example the excipient is polyethyleneglycol. In another example the excipient is precirol.

In another embodiment, the oral dosage form of the pharmaceutical composition includes a plasticizer. The plasticizer can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethyl citrate.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a rectal dosage form. In one embodiment, the rectal dosage form is an enema. In another embodiment, the rectal dosage form is a suppository.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a vaginal dosage form. In one embodiment, the vaginal dosage form is a suppository. In another embodiment, the vaginal dosage form is a foam, cream, or gel.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a pulmonary or nasal dosage form. In one embodiment, the iRNA agent is incorporated into a particle, e.g., a macroparticle, e.g., a microsphere. The particle can be produced by spray drying, lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination thereof. The microsphere can be formulated as a suspension, a powder, or an implantable solid.

In one aspect, the invention features a spray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition suitable for inhalation by a subject, including: (a) a therapeutically effective amount of a iRNA agent suitable for treating a condition in the subject by inhalation; (b) a pharmaceutically acceptable excipient selected from the group consisting of carbohydrates and amino acids; and (c) optionally, a dispersibility-enhancing amount of a physiologically-acceptable, water-soluble polypeptide.

In one embodiment, the excipient is a carbohydrate. The carbohydrate can be selected from the group consisting of monosaccharides, disaccharides, trisaccharides, and polysaccharides. In a preferred embodiment the carbohydrate is a monosaccharide selected from the group consisting of dextrose, galactose, mannitol, D-mannose, sorbitol, and sorbose. In another preferred embodiment the carbohydrate is a disaccharide selected from the group consisting of lactose, maltose, sucrose, and trehalose.

In another embodiment, the excipient is an amino acid. In one embodiment, the amino acid is a hydrophobic amino acid. In a preferred embodiment the hydrophobic amino acid is selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. In yet another embodiment the amino acid is a polar amino acid. In a preferred embodiment the amino acid is selected from the group consisting of arginine, histidine, lysine, cysteine, glycine, glutamine, serine, threonine, tyrosine, aspartic acid and glutamic acid.

In one embodiment, the dispersibility-enhancing polypeptide is selected from the group consisting of human serum albumin, α-lactalbumin, trypsinogen, and polyalanine.

In one embodiment, the spray-dried iRNA agent composition includes particles having a mass median diameter (MMD) of less than 10 microns. In another embodiment, the spray-dried iRNA agent composition includes particles having a mass median diameter of less than 5 microns. In yet another embodiment the spray-dried iRNA agent composition includes particles having a mass median aerodynamic diameter (MMAD) of less than 5 microns.

In certain other aspects, the invention provides kits that include a suitable container containing a pharmaceutical formulation of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). In certain embodiments the individual components of the pharmaceutical formulation may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation separately in two or more containers, e.g., one container for an iRNA agent preparation, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device.

In another aspect, the invention features a device, e.g., an implantable device, wherein the device can dispense or administer a composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), e.g., a iRNA agent that silences an endogenous transcript. In one embodiment, the device is coated with the composition. In another embodiment the iRNA agent is disposed within the device. In another embodiment, the device includes a mechanism to dispense a unit dose of the composition. In other embodiments the device releases the composition continuously, e.g., by diffusion. Exemplary devices include stents, catheters, pumps, artificial organs or organ components (e.g., artificial heart, a heart valve, etc.), and sutures.

As used herein, the term “crystalline” describes a solid having the structure or characteristics of a crystal, i.e., particles of three-dimensional structure in which the plane faces intersect at definite angles and in which there is a regular internal structure. The compositions of the invention may have different crystalline forms. Crystalline forms can be prepared by a variety of methods, including, for example, spray drying.

The invention is further illustrated by the following examples, which should not be construed as further limiting.

EXAMPLES Example 1 Inhibition of Endogenous ApoM Gene Expression in Mice

Apolipoprotein M (ApoM) is a human apolipoprotein predominantly present in high-density lipoprotein (HDL) in plasma. ApoM is reported to be expressed exclusively in liver and in kidney (Xu N et al., Biochem J Biol Chem 1999 Oct. 29; 274(44):31286-90). Mouse ApoM is a 21 kD membrane associated protein, and, in serum, the protein is associated with HDL particles. ApoM gene expression is regulated by the transcription factor hepatocyte nuclear factor 1 alpha (Hnf-1α), as Hnf-1α^(−/−) mice are ApoM deficient. In humans, mutations in the HNF-1 alpha gene represent a common cause of maturity-onset diabetes of the young (MODY).

A variety of test iRNAs were synthesized to target the mouse ApoM gene. This gene was chosen in part because of its high expression levels and exclusive activity in the liver and kidney.

Three different classes of dsRNA agents were synthesized, each class having different modifications and features at the 5′ and 3′ ends, see Table 4.

TABLE 4 Targeted ORF's  5 The23mer: AAGTTTGGGCAGCTCTGCTCT (SEQ ID NO: 6708) 19 The23mer: AAGTGGACATACCGATTGACT (SEQ ID NO: 6709) 25 The23mer: AACTCAGAACTGAAGGGCGCC (SEQ ID NO: 6710) 27 The23mer: AAGGGCGCCCAGACATGAAAA (SEQ ID NO: 6711) 3′-UTR (beginning at 645) 42: AAGATAGGAGCCCAGCTTCGA (SEQ ID NO: 6712) Class I 21-nt iRNAs, t, deoxythymidine; p, phosphate pGUUUGGGCAGCUCUGCUCUtt (SEQ ID NO: 6712) #1 pAGAGCAGAGCUGCCCAAACtt (SEQ ID NO: 6713) pGUGGACAUACCGAUUGACUtt (SEQ ID NO: 6714) #2 pAGUCAAUCGGUAUGUCCACtt (SEQ ID NO: 6715) pCUCAGAACUGAAGGGCGCCtt (SEQ ID NO: 6716) #3 pGGCGCCCUUCAGUUCUGAGtt (SEQ ID NO: 6717) pGAUAGGAGCCCAGCUUCGAtt (SEQ ID NO: 6718) #4 pUCGAAGCUGGGCUCCUAUCtt (SEQ ID NO: 6719) Class II 21-nt iRNAs, t, deoxythymidine; p, phosphate; ps, thiophosphate pGUUUGGGCAGCUCUGCUCpsUpstpst (SEQ ID NO: 6720) #11 pAGAGCAGAGCUGCCCAAApsCpstpst (SEQ ID NO: 6721) pGUGGACAUACCGAUUGACpsUpstpst (SEQ ID NO: 6722) #13 pAGUCAAUCGGUAUGUCCApsCpstpst (SEQ ID NO: 6723) pCUCAGAACUGAAGGGCGCpsCpstpst (SEQ ID NO: 6724) #15 pGGCGCCCUUCAGUUCUGApsGpstpst (SEQ ID NO: 6725) pGAUAGGAGCCCAGCUUCGpsApstpst (SEQ ID NO: 6726) #17 pUCGAAGCUGGGCUCCUAUpsCpstpst (SEQ ID NO: 6727) Class III 23-nt antisense, 21-nt sense, blunt-ended 5′-as GUUUGGGCAGCUCUGCUCUCU (SEQ ID NO: 6728) #19 AGAGAGCAGAGCUGCCCAAACUU (SEQ ID NO: 6729) GUGGACAUACCGAUUGACUGA (SEQ ID NO: 6730) #21 UCAGUCAAUCGGUAUGUCCACUU (SEQ ID NO: 6731) CUCAGAACUGAAGGGCGCCCA (SEQ ID NO: 6732) #23 PUGGGCGCCCUUCAGUUCUGAGUU (SEQ ID NO: 6733) GAUAGGAGCCCAGCUUCGAGU (SEQ ID NO: 6734) #25 ACUCGAAGCUGGGCUCCUAUCUU (SEQ ID NO: 6735)

Class I dsRNAs consisted of 21 nucleotide paired sense and antisense strands. The sense and antisense strands were each phosphorylated at their 5′ ends. The double stranded region was 19 nucleotides long and consisted of ribonucleotides. The 3′ end of each strand created a two nucleotide overhang consisting of two deoxyribonucleotide thymidines. See constructs #1-4 in Table 4.

Class II dsRNAs were also 21 nucleotides long, with a 19 nucleotide double strand region. The sense and antisense strands were each phosphorylated at their 5′ ends. The three 3′ terminal nucleotides of the sense and antisense strands were phosphorothioate deoxyribonucleotides, and the two terminal phosphorothioate thymidines were unpaired, creating a 3′ overhang region at each end of the iRNA molecule. See constructs 11, 13, 15, and 17 in Table 4.

Class III dsRNAs included a 23 ribonucleotide antisense strand and a 21 ribonucleotide sense strand, to form a construct having a blunt 5′ and a 3′ overhang region. See constructs 19, 21, 23, and 25 in Table 4.

Within each of the three classes of iRNAs, the four dsRNA molecules were designed to target four different regions of the ApoM transcript. dsRNAs 1, 11, and 19 targeted the 5′ end of the open reading frame (ORF). dsRNAs 2, 13, and 21, and 3, 15, and 23, targeted two internal regions (one 5′ proximal and one 3′ proximal) of the ORF, and the 4, 17, and 25 iRNA constructs targeted to a region of the 3′ untranslated sequence (3′ UTS) of the ApoM mRNA. This is summarized in Table 5.

TABLE 5 iRNA molecules targeted to mouse ApoM iRNA iRNA targeted iRNA targeted iRNA targeted targeted to 5′ end of to middle ORF to middle ORF to ORF (5′ proximal) (3′ proximal) 3′UTS Class I 1 2 3 4 Class II 11 13 15 17 Class III 19 21 23 25

CD1 mice (6-8 weeks old, ˜35 g) were administered one of the test iRNAs in PBS solution. Two hundred micrograms of iRNA in a volume of solution equal to 10% body weight (˜5.7 mg iRNA/kg mouse) was administered by the method of high pressure tail vein injection, over a 10-20 sec. time interval. After a 24 h recovery period, a second injection was performed using the same dose and mode of administration as the first injection, and following another 24 h, a third and final injection was administered, also using the same dose and mode of administration. After a final 24 h recovery, the mouse was sacrificed, serum was collected and the liver and kidney harvested to assay for an affect on ApoM gene expression. Expression was monitored by quantitative RT-PCR and Western blot analyses. This experiment was repeated for each of the iRNAs listed in table 4.

Class I iRNAs did not alter ApoM RNA levels in mice, as indicated by quantitative RT-PCR. This is in contrast to the effect of these iRNAs in cultured HepG2 cells. Cells cotransfected with a plasmid expressing exogenous ApoM RNA under a CMV promoter and a class I iRNA demonstrated a 25% or greater reduction in ApoM RNA concentrations as compared to control transfections. The iRNA molecules 1, 2 and 3 each caused a 75% decrease in exogenous ApoM mRNA levels.

Class II iRNAs reduced liver and kidney ApoM mRNA levels by ˜30-85%. The iRNA molecule “13” elicited the most dramatic reduction in mRNA levels; quantitative RT-PCR indicated a decrease of about 85% in liver tissue. Serum ApoM protein levels were also reduced as was evidenced by Western blot analysis. The iRNAs 11, 13 and 15, reduced protein levels by about 50%, while iRNA 17 had the mildest effect, reducing levels only by ˜15-20%.

Class III iRNAs (constructs 19, 21, and 23) reduced serum Apo levels by ˜40-50%.

To determine the effect of dosage on iRNA mediated ApoM inhibition, the experiment described above was repeated with three injections of 50 μg iRNA “11” (˜1.4 mg iRNA/kg mouse). This lower dosage of iRNA resulted in a reduction of serum ApoM levels of about 50%. This is compared with the reduction seen with the 200 μg injections, which reduced serum levels by 25-45%. These results indicated the lower dosage amounts of iRNAs were effective.

In an effort to increase iRNA uptake by cells, iRNAs were precomplexed with lipofectamine prior to tail vein injections. ApoM protein levels were about 50% of wildtype levels in mice injected with iRNA “11” when the molecules were preincubated with lipofectamine; ApoM levels were also about 50% of wildtype when mice were injected with iRNA “11” that was not precomplexed with lipofectamine.

These experiments revealed that modified iRNAs can greatly influence RNAi-mediated gene silencing. As demonstrated herein, modifications including phosphorothioate nucleotides are particularly effective at decreasing target protein levels.

Example 2 apoB Protein as a Therapeutic Target for Lipid-Based Diseases

Apolipoprotein B (apoB) is a candidate target gene for the development of novel therapies for lipid-based diseases.

Methods described herein can be used to evaluate the efficacy of a particular siRNA as a therapeutic tool for treating lipid metabolism disorders resulting elevated apoB levels. Use of siRNA duplexes to selectively bind and inactivate the target apoB mRNA is an approach to treat these disorders.

Two Approaches:

i) Inhibition of apoB in ex-vivo models by transfecting siRNA duplexes homologous to human apoB mRNA in a human hepatoma cell line (Hep G2) and monitor the level of the protein and the RNA using the Western blotting and RT-PCR methods, respectively. siRNA molecules that efficiently inhibit apoB expression will be tested for similar effects in vivo.

ii) In vivo trials using an apoB transgenic mouse model (apoB100 Transgenic Mice, C57BL/6NTac-TgN (APOB100), Order Model #'s:1004-T (hemizygotes), B6 (control)). siRNA duplexes are designed to target apoB-100 or CETP/apoB double transgenic mice which express both cholesteryl ester transfer protein (CETP) and apoB. The effect of the siRNA on gene expression in vivo can be measured by monitoring the HDL/LDL cholesterol level in serum. The results of these experiments would indicate the therapeutic potential of siRNAs to treat lipid-based diseases, including hypercholesterolemia, HDL/LDL cholesterol imbalance, familial combined hyperlipidemia, and acquired hyperlipidemia.

Background

Fats, in the form of triglycerides, are ideal for energy storage because they are highly reduced and anhydrous. An adipocyte (or fat cell) consists of a nucleus, a cell membrane, and triglycerides, and its function is to store triglycerides.

The lipid portion of the human diet consists largely of triglycerides and cholesterol (and its esters). These must be emulsified and digested to be absorbed. Specifically, fats (triacylglycerols) are ingested. Bile (bile acids, salts, and cholesterol), which is made in the liver, is secreted by the gall bladder. Pancreatic lipase digests the triglycerides to fatty acids, and also digests di-, and mono-acylglycerols, which are absorbed by intestinal epithelial cells and then are resynthesized into triacylglycerols once inside the cells. These triglycerides and some cholesterols are combined with apolipoproteins to produce chylomicrons.

Chylomicrons consist of approximately 95% triglycerides. The chylomicrons transport fatty acids to peripheral tissues. Any excess fat is stored in adipose tissue.

Lipid transport and clearance from the blood into cells, and from the cells into the blood and the liver, is mediated by the lipoprotein transport proteins. This class of approximately 17 proteins can be divided into three groups: Apolipoproteins, lipoprotein processing proteins, and lipoprotein receptors.

Apolipoproteins coat lipoprotein particles, and include the A-I, A-II, A-IV, B, CI, CII, CIII, D, E, Apo(a) proteins. Lipoprotein processing proteins include lipoprotein lipase, hepatic lipase, lecithin cholesterol acyltransferase and cholesterol ester transfer protein. Lipoprotein receptors include the low density lipoprotein (LDL) receptor, chylomicron-remnant receptor (the LDL receptor like protein or LDL receptor related protein—LRP) and the scavenger receptor.

Lipoprotein Metabolism

Since the triglycerides, cholesterol esters, and cholesterol absorbed into the small intestine are not soluble in aqueous medium, they must be combined with suitable proteins (apolipoproteins) in order to prevent them from forming large oil droplets. The resulting lipoproteins undergo a type of metabolism as they pass through the bloodstream and certain organs (notably the liver).

Also synthesized in the liver is high density lipoprotein (HDL), which contains the apoproteins A-1, A-2, C-1, and D; HDL collects cholesterol from peripheral tissues and blood vessels and returns it to the liver. LDL is taken up by specific cell surface receptors into an endosome, which fuses with a lysosome where cholesterol ester is converted to free cholesterol. The apoproteins (including apo B-100) are digested to amino acids. The receptor protein is recycled to the cell membrane.

The free cholesterol formed by this process has two fates. First, it can move to the endoplasmic reticulum (ER), where it can inhibit HMG-CoA reductase, the synthesis of HMG-CoA reductase, and the synthesis of cell surface receptors for LDL. Also in the ER, cholesterol can speed up the degradation of HMG-CoA reductase. The free cholesterol can also be converted by acyl-CoA and acyl transferase (ACAT) to cholesterol esters, which form oil droplets.

ApoB is the major apolipoprotein of chylomicrons of very low density lipoproteins (VLDL, which carry most of the plasma triglyceride) and low density lipoprotein (LDL, which carry most of the plasma cholesterol). ApoB exists in human plasma in two isoforms, apoB-48 and apoB-100.

ApoB-100 is the major physiological ligand for the LDL receptor. The ApoB precursor has 4563 amino acids, and the mature apoB-100 has 4536 amino acid residues. The LDL-binding domain of ApoB-100 is proposed to be located between residues 3129 and 3532. ApoB-100 is synthesized in the liver and is required for the assembly of very low density lipoproteins VLDL and for the preparation of apoB-100 to transport triglycerides (TG) and cholesterol from the liver to other tissues. ApoB-100 does not interchange between lipoprotein particles, as do the other lipoproteins, and it is found in IDL and LDL particles. After the removal of apolipoproteins A, E and C, apoB is incorporation into VLDL by hepatocytes. ApoB-48 is present in chylomicrons and plays an essential role in the intestinal absorption of dietary fats. ApoB-48 is synthesized in the small intestine. It comprises the N-terminal 48% of apoB-100 and is produced by a posttranscriptional apoB-100 mRNA editing event at codon 2153 (C to U). This editing event is a product of the apoBEC-1b enzyme, which is expressed in the intestine. This editing event creates a stop codon instead of a glutamine codon, and therefore apoB-48, instead of apoB-100 is expressed in the intestine (apoB-100 is expressed in the liver).

There is also strong evidence that plasma apoB levels may be a better index of the risk of coronary artery disease (CAD) than total or LDL cholesterol levels. Clinical studies have demonstrated the value of measuring apoB in hypertriglyceridemic, hypercholesterolemic and normalipidemic subjects.

TABLE 6 Reference Range Lipid level in the Blood Lipid Range (mmols/L) Plasma Cholesterol 3.5-6.5 Low density lipoprotein 1.55-4.4  Very low density lipoprotein 0.128-0.645 High density lipoprotein/triglycerides 0.5-2.1 Total lipid 4.0-10 g/L Molecular Genetics of Lipid Metabolism in Both Humans and Induced Mutant Mouse Models

Elevated plasma levels of LDL and apoB are associated with a higher risk for atherosclerosis and coronary heart disease, a leading cause of mortality. ApoB is the mandatory constituent of LDL particles. In addition to its role in lipoprotein metabolism, apoB has also been implicated as a factor in male infertility and fetal development. Furthermore, two quantitative trait loci regulating plasma apoB levels have been discovered, through the use of transgenic mouse models. Future experiments will facilitate the identification of human orthologous genes encoding regulators of plasma apoB levels. These loci are candidate therapeutic targets for human disorders characterized by altered plasma apoB levels. Such disorders include non-apoB linked hypobetalipoproteinemia and familial combined hyperlipidemia. The identification of these genetic loci would also reveal possible new pathways involved in the regulation of apoB secretion, potentially providing novel sites for pharmacological therapy.

Diseases and Clinical Pharmacology

Familial combined hyperlipemia (FCHL) affects an estimated one in 10 Americans. FCHL can cause premature heart disease.

Familial Hypercholesterolemia (High Level of apo B)

A common genetic disorder of lipid metabolism. Familial hypercholesterolemia is characterized by elevated serum TC in association with xanthelasma, tendon and tuberous xanthomas, accelerated atherosclerosis, and early death from myocardial infarction (MI). It is caused by absent or defective LDL cell receptors, resulting in delayed LDL clearance, an increase in plasma LDL levels, and an accumulation of LDL cholesterol in macrophages over joints and pressure points, and in blood vessels.

Atherosclerosis (High Level of apo B)

Atherosclerosis develops as a deposition of cholesterol and fat in the arterial wall due to disturbances in lipid transport and clearance from the blood into cells and from the cells to blood and the liver.

Clinical studies have demonstrated that elevation of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and apoB-100 promote human atherosclerosis. Similarly, decreased levels of high-density lipoprotein cholesterol (HDL-C) are associated with the development of atherosclerosis.

ApoB may be factor in the genetic cause of high cholesterol.

The Risk of Coronary Artery Disease (CAD) (High Level of apo B)

Cardiovascular disease, including coronary heart disease and stroke, is a leading cause of death and disability. The major risk factors include age, gender, elevated low-density lipoprotein cholesterol blood levels, decreased high-density lipoprotein cholesterol levels, cigarette smoking, hypertension, and diabetes. Emerging risk factors include elevated lipoprotein (a), remnant lipoproteins, and C reactive protein. Dietary intake, physical activity and genetics also impact cardiovascular risk. Hypertension and age are the major risk factors for stroke.

Abetalipoproteinemia, an inherited human disease characterized by a near-complete absence of apoB-containing lipoproteins in the plasma, is caused by mutations in the gene for microsomal triglyceride transfer protein (MTP).

Model for Human Atherosclerosis (Lipoprotein A Transgenic Mouse)

Numerous studies have demonstrated that an elevated plasma level of lipoprotein(a) (Lp(a)) is a major independent risk factor for coronary heart disease (CHD). Current therapies, however, have little or no effect on apo(a) levels and the homology between apo(a) and plasminogen presents barriers to drug development. Lp(a) particles consist of apo(a) and apoB-100 proteins, and they are found only in primates and the hedgehog. The development of LPA transgenic mouse requires the creation of animals that express both human apoB and apo(a) transgenes to achieve assembly of LP(a). An atherosclerosis mouse model would facilitate the study of the disease process and factors influencing it, and further would facilitate the development of therapeutic or preventive agents. There are several strategies for gene-oriented therapy. For example, the missing or non-functional gene can be replaced, or unwanted gene activity can be inhibited.

Model for Lipid Metabolism and Atherosclerosis

DNX Transgenic Sciences has demonstrated that both CETP/ApoB and ApoB transgenic mice develop atherosclerotic plaques.

Model for apoB-100 Overexpression

The apoB-100 transgenic mice express high levels of human apoB-100. They consequently demonstrate elevated serum levels of LDL cholesterol. After 6 months on a high-fat diet, the mice develop significant foam cell accumulation under the endothelium and within the media, as well as cholesterol crystals and fibrotic lesions.

Model for Cholesteryl Ester Transfer Protein Over Expression

The apoB-100 transgenic mice express the human enzyme, CETP, and consequently demonstrate a dramatically reduced level of serum HDL cholesterol.

Model for apoB-100 and CETP Overexpression

The apoB-100 transgenic mice express both CETP and apoB-100, resulting in mice with a human like serum HDL/LDL distribution. Following 6 months on a high-fat diet these mice develop significant foam cell accumulation underlying the endothelium and within the media, as well as cholesterol crystals and fibrotic lesions.

ApoB100 Transgenic Mice (Order Model #'s:1004-T (Hemizygotes), B6 (Control))

These mice express high levels of human apoB-100, resulting in mice with elevated serum levels of LDL cholesterol. These mice are useful in identifying and evaluating compounds to reduce elevated levels of LDL cholesterol and the risk of atherosclerosis. When fed a high fat cholesterol diet, these mice develop significant foam cell accumulation underlay the endothelium and within the media, and have significantly more complex atherosclerotic lesions than control animals.

Double Transgenic Mice, CETP/ApoB100 (Order Model #: 1007-TT)

These mice express both CETP and apoB-100, resulting in a human-like serum HDL/LDL distribution. These mice are useful for evaluating compounds to treat hypercholesterolemia or HDL/LDL cholesterol imbalance to reduce the risk of developing atherosclerosis. When fed a high fat high cholesterol diet, these mice develop significant foam cell accumulation underlying the endothelium and within the media, and have significantly more complex atherosclerotic lesions than control animals.

ApoE Gene Knockout Mouse

Homozygous apoE knockout mice exhibit strong hypercholesterolemia, primarily due to elevated levels of VLDL and IDL caused by a defect in lipoprotein clearance from plasma. These mice develop atherosclerotic lesions which progress with age and resemble human lesions (Zhang et al., Science 258:46-71, 1992; Plump et al., Cell 71:343-353, 1992; Nakashima et al., Arterioscler Thromp. 14:133-140, 1994; Reddick et al., Arterioscler Tromb. 14:141-147, 1994). These mice are a promising model for studying the effect of diet and drugs on atherosclerosis.

Low density lipoprotein receptor (LDLR) mediates lipoprotein clearance from plasma through the recognition of apoB and apoE on the surface of lipoprotein particles. Humans, who lack or have a decreased number of the LDL receptors, have familial hypercholesterolemia and develop CHD at an early age.

ApoE Knockout Mice (Order Model #: APOE-M)

The apoE knockout mouse was created by gene targeting in embryonic stem cells to disrupt the apoE gene. ApoE, a glycoprotein, is a structural component of very low density lipoprotein (VLDL) synthesized by the liver and intestinally synthesized chylomicrons. It is also a constituent of a subclass of high density lipoproteins (HDLs) involved in cholesterol transport activity among cells. One of the most important roles of apoE is to mediate high affinity binding of chylomicrons and VLDL particles that contain apoE to the low density lipoprotein (LDL) receptor. This allows for the specific uptake of these particles by the liver which is necessary for transport preventing the accumulation in plasma of cholesterol-rich remnants. The homozygous inactivation of the apoE gene results in animals that are devoid of apoE in their sera. The mice appear to develop normally, but they exhibit five times the normal serum plasma cholesterol and spontaneous atherosclerotic lesions. This is similar to a disease in people who have a variant form of the apoE gene that is defective in binding to the LDL receptor and are at risk for early development of atherosclerosis and increased plasma triglyceride and cholesterol levels. There are indications that apoE is also involved in immune system regulation, nerve regeneration and muscle differentiation. The apoE knockout mice can be used to study the role of apoE in lipid metabolism, atherogenesis, and nerve injury, and to investigate intervention therapies that modify the atherogenic process.

Apoe4 Targeted Replacement Mouse (Order Model #: 001549-M)

ApoE is a plasma protein involved in cholesterol transport, and the three human isoforms (E2, E3, and E4) have been associated with atherosclerosis and Alzheimer's disease. Gene targeting of 129 ES cells was used to replace the coding sequence of mouse apoE with human APOE4 without disturbing the murine regulatory sequences. The E4 isoform occurs in approximately 14% of the human population and is associated with increased plasma cholesterol and a greater risk of coronary artery disease. The Taconic apoE4 Targeted Replacement model has normal plasma cholesterol and triglyceride levels, but altered quantities of different plasma lipoprotein particles. This model also has delayed plasma clearance of cholesterol-rich lipoprotein particles (VLDL), with only half the clearance rate seen in the apoE3 Targeted Replacement model. Like the apoE3 model, the apoE4 mice develop altered plasma lipoprotein values and atherosclerotic plaques on an atherogenic diet. However, the atherosclerosis is more severe in the apoE4 model, with larger plaques and cholesterol apoE and apoB-48 levels twice that seen in the apoE3 model. The Taconic apoE4 Targeted Replacement model, along with the apoE2 and apoE3 Targeted Replacement Mice, provide an excellent tool for in vivo study of the human apoE isoforms.

CETP Transgenic Mice (Order Model #: 1003-T)

These animals express the human plasma enzyme, CETP, resulting in mice with a dramatic reduction in serum HDL cholesterol. The mice can be useful in identifying and evaluating compounds that increase the levels of HDL cholesterol for reducing the risk of developing atherosclerosis

Transgene/Promoter: Human Apolipoprotein A-I

These mice produce mouse HDL cholesterol particles that contain human apolipoprotein A-I. Transgenic expression is life-long in both sexes (Biochemical Genetics and Metabolism Laboratory, Rockefeller University, NY City).

A Mouse Model for Abetalipoproteinemia

Abetalipoproteinemia, an inherited human disease characterized by a near-complete absence of apoB-containing lipoproteins in the plasma, is caused by mutations in the gene for microsomal triglyceride transfer protein (MTP). Gene targeting was used to knock out the mouse MTP gene (Mttp). In heterozygous knockout mice (Mttp^(+/−)), the MTP mRNA, protein, and activity levels were reduced by 50% in both liver and intestine. Recent studies with heterozygous MTP knockout mice have suggested that half-normal levels of MTP in the liver reduce apoB secretion. They hypothesized that reduced apoB secretion in the setting of half-normal MTP levels might be caused by a reduced MTP:apoB ratio in the endoplasmic reticulum, which would reduce the number of apoB-MTP interactions. If this hypothesis were true, half-normal levels of MTP might have little impact on lipoprotein secretion in the setting of half-normal levels of apoB synthesis (since the ratio of MTP to apoB would not be abnormally low) and might cause an exaggerated reduction in lipoprotein secretion in the setting of apoB overexpression (since the ratio of MTP to apoB would be even lower). To test this hypothesis, they examined the effects of heterozygous MTP deficiency on apoB metabolism in the setting of normal levels of apoB synthesis, half-normal levels of apoB synthesis (heterozygous Apob deficiency), and increased levels of apoB synthesis (transgenic overexpression of human apoB). Contrary to their expectations, half-normal levels of MTP reduced plasma apoB-100 levels to the same extent (˜25-35%) at each level of apoB synthesis. In addition, apoB secretion from primary hepatocytes was reduced to a comparable extent at each level of apoB synthesis. Thus, these results indicate that the concentration of MTP within the endoplasmic reticulum, rather than the MTP:apoB ratio, is the critical determinant of lipoprotein secretion. Finally, heterozygosity for an apoB knockout mutation was found to lower plasma apoB-100 levels more than heterozygosity for an MTP knockout allele. Consistent with that result, hepatic triglyceride accumulation was greater in heterozygous apoB knockout mice than in heterozygous MTP knockout mice. Cre/loxP tissue-specific recombination techniques were also used to generate liver-specific Mttp knockout mice. Inactivation of the Mttp gene in the liver caused a striking reduction in very low density lipoprotein (VLDL) triglycerides and large reductions in both VLDL/low density lipoproteins (LDL) and high density lipoprotein cholesterol levels. Histologic studies in liver-specific knockout mice revealed moderate hepatic steatosis. Currently being tested is the hypothesis that accumulation of triglycerides in the liver renders the liver more susceptible to injury by a second insult (e.g., lipopolysaccharide).

Human apo B (Apolipoprotein B) Transgene Mice Show apo B Locus May have a Causative Role Male Infertility

The fertility of apoB (apolipoprotein B) (+/−) mice was recorded during the course of backcrossing (to C57BL/6J mice) and test mating. No apparent fertility problem was observed in female apoB (+/−) and wild-type female mice, as was documented by the presence of vaginal plugs in female mice. Although apoB (+/−) mice mated normally, only 40% of the animals from the second backcross generation produced any offspring within the 4-month test period. Of the animals that produced progeny, litters resulted from <50% of documented matings. In contrast, all wild-type mice (6/6—i.e., 100%) tested were fertile. These data suggest genetic influence on the infertility phenotype, as a small number of male heterozygotes were not sterile. Fertilization in vivo was dramatically impaired in male apoB (+/−) mice. 74% of eggs examined were fertilized by the sperm from wild-type mice, whereas only 3% of eggs examined were fertilized by the sperm from apoB (+/−) mice. The sperm counts of apoB (+/−) mice were mildly but significantly reduced compared with controls. However, the percentage of motile sperm was markedly reduced in the apoB (+/−) animals compared with that of the wild-type controls. Of the sperm from apoB (+/−) mice, 20% (i.e., 4.9% of the initial 20% motile sperm) remained motile after 6 hr of incubation, whereas 45% (i.e., 33.6% of the initial 69.5%) of the motile sperm retained motility in controls after this time. In vitro fertilization yielded no fertilized eggs in three attempts with apo B (+/−) mice, while wild-type controls showed a fertilization rate of 53%. However, sperm from apoB (+/−) mice fertilized 84% of eggs once the zona pellucida had been removed. Numerous sperm from apoB (+/−) mice were seen binding to zona-intact eggs. However, these sperm lost their motility when observed 4-6 hours after binding, showing that sperm from apoB (+/−) mice were unable to penetrate the zona pellucida but that the interaction between sperm and egg was probably not direct. Sperm binding to zona-free oocytes was abnormal. In the apoB (+/−) mice, sperm binding did not attenuate, even after pronuclei had clearly formed, suggesting that apoB deficiency results in abnormal surface interaction between the sperm and egg.

Knockout of the mouse apoB gene resulted in embryonic lethality in homozygotes, protection against diet-induced hypercholesterolemia in heterozygotes, and developmental abnormalities in mice.

Model of Insulin Resistance, Dyslipidemia & Overexpression of Human apoB

It was shown that the livers of apoB mice assemble and secrete increased numbers of VLDL particles.

Example 3 Treatment of Diabetes Type-2 with iRNA

Introduction

The regulation of hepatic gluconeogenesis is an important process in the adjustment of the blood glucose level. Pathological changes in the glucose production of the liver are a central characteristic in type-2-diabetes. For example, the fasting hyperglycemia observed in patients with type-2-diabetes reflects the lack of inhibition of hepatic gluconeogenesis and glycogenolysis due to the underlying insulin resistance in this disease. Extreme conditions of insulin resistance can be observed for example in mice with a liver-specific insulin receptor knockout (‘LIRKO’). These mice have an increased expression of the two rate-limiting gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and the glucose-6-phosphatase catalytic subunit (G6Pase). Insulin is known to repress both PEPCK and G6Pase gene expression at the transcriptional level and the signal transduction involved in the regulation of G6Pase and PEPCK gene expression by insulin is only partly understood. While PEPCK is involved in a very early step of hepatic gluconeogenesis (synthesis of phosphoenolpyruvate from oxaloacetate), G6Pase catalyzes the terminal step of both, gluconeogenesis and glycogenolysis, the cleavage of glucose-6-phosphate into phosphate and free glucose, which is then delivered into the blood stream.

The pharmacological intervention in the regulation of expression of PEPCK and G6Pase can be used for the treatment of the metabolic aberrations associated with diabetes. Hepatic glucose production can be reduced by an iRNA-based reduction of PEPCK and G6Pase enzymatic activity in subjects with type-2-diabetes.

Targets for iRNA

Glucose-6-phosphatase (G6Pase)

G6Pase mRNA is expressed principally in liver and kidney, and in lower amounts in the small intestine. Membrane-bound G6Pase is associated with the endoplasmic reticulum. Low activities have been detected in skeletal muscle and in astrocytes as well.

G6Pase catalyzes the terminal step in gluconeogenesis and glycogenolysis. The activity of the enzyme is several fold higher in diabetic animals and probably in diabetic humans. Starvation and diabetes cause a 2-3-fold increase in G6Pase activity in the liver and a 2-4-fold increase in G6Pase mRNA.

Phosphoenolpyruvate Carboxykinase (PEPCK)

Overexpression of PEPCK in mice results in symptoms of type-2-diabetes mellitus. PEPCK overexpression results in a metabolic pattern that increases G6Pase mRNA and results in a selective decrease in insulin receptor substrate (IRS)-2 protein, decreased phosphatidylinositol 3-kinase activity, and reduced ability of insulin to suppress gluconeogenic gene expression.

TABLE 7 Other targets to inhibit hepatic glucose production Target Comment FKHR good evidence for antidiabetic phenotype (Nakae et al., Nat Genetics 32: 245(2002) Glucagon Glucagon receptor Glycogen phosphorylase PGC-1 (PPAR-Gamma regulates the cAMP response (and Coactivator) probably the PKB/FKHR-regulation) on PEPCK/G6Pase Fructose-1,6-bisphosphatase Glucose-6-phospate translocator Glucokinase inhibitory regulatory protein Materials and Methods

Animals: BKS.Cg-m+/+Lepr db mice, which contain a point mutation in the leptin receptor gene are used to examine the efficacy of iRNA for the targets listed above.

BKS.Cg-m+/+Lepr db are available from the Jackson Laboratory (Stock Number 000642). These animals are obese at 3-4 weeks after birth, show elevation of plasma insulin at 10 to 14 days, elevation of blood sugar at 4 to 8 weeks, and uncontrolled rise in blood sugar. Exogenous insulin fails to control blood glucose levels and gluconeogenic activity increases.

The following numbers of male animals (age>12 weeks) would ideally be tested with the following iRNAs:

PEPCK, 2 sequences, 5 animals per sequence

-   -   G6Pase, 2 sequences, 5 animals per sequence     -   1 nonspecific sequence, 5 animals     -   1 control group (only injected, no siRNA), 5 animals     -   1 control group (not injected, no siRNA), 5 animals

Reagents: Necessary reagents would ideally include a Glucometer Elite XL (Bayer, Pittsburgh, Pa.) for glucose quantification, and an Insulin Radioimmunoassay (RIA) kit (Amersham, Piscataway, N.J.) for insulin quantitation

Assays:

G6P enzyme assays and PEPCK enzyme assays are used to measure the activity of the enzymes. Northern blotting is used to detect levels of G6Pase and PEPCK mRNA. Antibody-based techniques (e.g., immunoblotting, immunofluorescence) are used to detect levels of G6Pase and PEPCK protein. Glycogen staining is used to detect levels of glycogen in the liver. Histological analysis is performed to analyze tissues.

Gene Information:

G6Pase GenBank® No.: NM_(—)008061, Mus musculus glucose-6-phosphatase, catalytic (G6pc), mRNA 1 . . . 2259, ORF 83 . . . 1156;

GenBank® No: U00445, Mus musculus glucose-6-phosphatase mRNA, complete cds 1 . . . 2259, ORF 83 . . . 1156

GenBank® No: BC013448

PEPCK

GenBank® No: NM_(—)011044, Mus musculus phosphoenolpyruvate carboxykinase 1, cytosolic (Pck1), mRNA.1 . . . 2618, ORF 141 . . . 2009

GenBank® No: AF009605.1

Administration of iRNA:

iRNA corresponding to the genes described above would be administered to mice with hydrodynamic injection. One control group of animals would be treated with Metformin as a positive control for reduction in hepatic glucose levels.

Experimental Protocol

Mice would be housed in a facility in which there is light from 7:00 AM to 7:00 PM. Mice would be fed ad libidum from 7:00 PM to 7:00 AM and fast from 7:00 AM to 7:00 PM.

Day 0: 7:00 PM: Approximately 100 μl blood would be drawn from the tail. Serum would be isolated to measure glucose, insulin, HbA1c (EDTA-blood), glucagon, FFAs, lactate, corticosterone, serum triglycerides.

Day 1-7: Blood glucose would be measured daily at 8:00 AM and 6:00 PM (approx. 3-5 μl; measured with a Haemoglucometer)

Day 8: Blood glucose would be measured daily at 8:00 AM and 6:00 PM. iRNA would be injected between 10:00 AM and 2:00 PM

Day 9-20: Blood glucose would be measured daily at 8:00 AM and 6:00 PM.

Day 21: Mice would be sacrificed after 10 hours of fasting.

Blood would be isolated. Glucose, insulin, HbA1c (EDTA-blood), glucagon, FFAs, lactate, corticosterone, serum triglycerides would be measured. Liver tissue would be isolated for histology, protein assays, RNA assays, glycogen quantitation, and enzyme assays.

Example 4 Inhibition of Glucose-6-Phosphatase iRNA In Vivo

iRNA targeted to the Glucose-6-Phosphatase (G6P) gene was used to examine the effects of inhibition of G6P expression on glucose metabolism in vivo.

Female mice, 10 weeks of age, strain BKS.Cg-m+/+Lepr db (The Jackson Laboratory) were used for in vivo analysis of enzymes of the hepatic glucose production. Mice were housed under conditions where it was light from 6:30 am to 6:30 pm. Mice were fed (ad libidum) during the night period and fasted during the day period.

On day 1, approximately 100 μl of blood was collected from test animals by puncturing the retroorbital plexus. On days 1-7, blood glucose was measured in blood obtained from tail veins (approximately 3-5 μl) using a Glucometer (Elite XL, Bayer). Blood glucose was sampled daily at 8 am and 6 pm.

On day 7 at approximately 2 pm, GL3 plasmid (10 μg) and siRNAs (100 μg G6Pase specific, Renilla nonspecific or no siRNA control) were delivered to animals using hydrodynamic coinjection.

On day 8, GL3 expression was analyzed by injection of luceferin (3 mg) after anaesthesia with avertin and imaging. This was done to control for successful hydrodynamic delivery.

On days 8-10, blood glucose was measured in blood obtained from tail veins (approximately 3-5 ml) using a Glucometer (Elite XL, Bayer).

On day 10, mice were sacrificed after 10 hours of fasting. Blood and liver were isolated from sacrificed animals.

Results: Coinjection of GL3 plasmid and G6Pase iRNA (G6P4) reduced blood glucose levels for the short term. Coinjection of GL3 plasmid and Renilla nonspecific iRNA had no effect on blood glucose levels.

Example 5 Selected Palindromic Sequences

Tables 8-13 below provide selected palindromic sequences from the following genes: human ApoB, human glucose-6-phosphatase, rat glucose-6-phosphatase, β-catenin, and hepatitis C virus (HCV).

TABLE 8 Selected palindromic sequences from human ApoB Start End Start End Source Index Index Match Index Index # B SEQ ID NO: 1 ggccattccagaagggaag 509 528 SEQ ID NO: 1004 cttccgttctgtaatggcc 5795 5814 1 9 SEQ ID NO: 2 tgccatctcgagagttcca 4099 4118 SEQ ID NO: 1005 tggaactctctccatggca 10878 10895 1 8 SEQ ID NO: 3 catgtcaaacactttgtta 7058 7075 SEQ ID NO: 1006 taacaaattccttgacatg 7358 7377 1 8 SEQ ID NO: 4 tttgttataaatcttattg 7068 7087 SEQ ID NO: 1007 caataagatcaatagcaaa 8990 9009 1 8 SEQ ID NO: 5 tctggaaaagggtcatggc 8880 8899 SEQ ID NO: 1008 tccatgtcccatttacaga 11356 11375 1 8 SEQ ID NO: 6 cagctcttgttcaggtcca 10900 10919 SEQ ID NO: 1009 tggacctgcaccaaagctg 13952 13971 1 8 SEQ ID NO: 7 ggaggttccccagctctgc 356 375 SEQ ID NO: 1010 gcagccctgggaaaactcc 6447 6466 1 7 SEQ ID NO: 8 ctgttttgaagactctcca 1081 1100 SEQ ID NO: 1011 tggagggtagtcataacag 10327 10348 1 7 SEQ ID NO: 9 agtggctgaaacgtgtgca 1297 1316 SEQ ID NO: 1012 tgcagagctttctgccact 13508 13527 1 7 SEQ ID NO: 10 ccaaaatagaagggaatct 2068 2087 SEQ ID NO: 1013 agattcctttgccttttgg 4000 4019 1 7 SEQ ID NO: 11 tgaagagaagattgaattt 3820 3839 SEQ ID NO: 1014 aaattctcttttcttttca 9212 9231 1 7 SEQ ID NO: 12 agtggtggcaacaccagca 4230 4249 SEQ ID NO: 1015 tgctagtgaggccaacact 10649 10668 1 7 SEQ ID NO: 13 aaggctccacaagtcatca 5950 5969 SEQ ID NO: 1016 tgatgatatctggcacctt 10724 10743 1 7 SEQ ID NO: 14 gtcagccaggtttatagca 7725 7744 SEQ ID NO: 1017 tgctaagaaccttactgac 7781 7800 1 7 SEQ ID NO: 15 tgatatctggaaccttgaa 10727 10748 SEQ ID NO: 1018 ttcactgttcctgaaatca 7863 7882 1 7 SEQ ID NO: 16 gtcaagttgagcaatttct 13423 13442 SEQ ID NO: 1019 agaaaaggcacaccttgac 11072 11091 1 7 SEQ ID NO: 17 atccagatggaaaagggaa 13480 13499 SEQ ID NO: 1020 ttccaatttccctgtggat 3880 3699 1 7 SEQ ID NO: 18 atttgtttgtcaaagaagt 4543 4562 SEQ ID NO: 1021 acttcagagaaatacaaat 11401 11420 4 6 SEQ ID NO: 19 ctggaaaatgtcagcctgg 204 223 SEQ ID NO: 1022 ccagacttccgtttaccag 8235 8254 2 6 SEQ ID NO: 20 accaggaggttcttcttca 1729 1748 SEQ ID NO: 1023 tgaagtgtagtctcctggt 5089 5108 2 6 SEQ ID NO: 21 aaagaagttctgaaagaat 1956 1975 SEQ ID NO: 1024 attccatcacaaatccttt 9661 9680 2 6 SEQ ID NO: 22 gctacagcttatggctcca 3570 3589 SEQ ID NO: 1025 tggatctaaatgcagtagc 11623 11842 2 6 SEQ ID NO: 23 atcaatattgatcaatttg 6414 6433 SEQ ID NO: 1026 caaagaagtcaagattgat 4553 4572 2 6 SEQ ID NO: 24 gaattatcttttaaaacat 7326 7345 SEQ ID NO: 1027 atgtgttaacaaaatattc 11494 11513 2 6 SEQ ID NO: 25 cgaggcccgcgctgctggc 130 149 SEQ ID NO: 1028 gccagaagtgagatcctcg 3507 3526 1 6 SEQ ID NO: 26 acaactatgaggctgagag 271 290 SEQ ID NO: 1029 ctctgagcaacaaatttgt 10309 10328 1 6 SEQ ID NO: 27 gctgagagttccagtggag 282 301 SEQ ID NO: 1030 ctccatggcaaatgtcagc 10885 10904 1 6 SEQ ID NO: 28 tgaagaaaaccaagaactc 448 487 SEQ ID NO: 1031 gagtcattgaggttcttca 4929 4948 1 6 SEQ ID NO: 29 cctacttacatcctgaaca 558 577 SEQ ID NO: 1032 tgttcataagggaggtagg 12766 12785 1 6 SEQ ID NO: 30 ctacttacatcctgaacat 559 578 SEQ ID NO: 1033 atgttcataagggaggtag 12785 12784 1 6 SEQ ID NO: 31 gagacagaagaagccaagc 615 634 SEQ ID NO: 1034 gcttggttttgccagtctc 2459 2478 1 6 SEQ ID NO: 32 cactcactttaccgtcaag 671 690 SEQ ID NO: 1035 cttgaacacaaagtcagtg 6000 6019 1 6 SEQ ID NO: 33 ctgatcagcagcagccagt 822 841 SEQ ID NO: 1036 actgggaagtgcttatcag 5237 5256 1 6 SEQ ID NO: 34 actagacgctaagaggaag 854 873 SEQ ID NO: 1037 cttccccaaagagaccagt 2890 2909 1 6 SEQ ID NO: 35 agaggaagcatgtggcaga 865 884 SEQ ID NO: 1038 tctggcatttactttctct 5921 5940 1 6 SEQ ID NO: 36 tgaagactctccaggaact 1087 1106 SEQ ID NO: 1039 agttgaaggagactattca 7218 7236 1 6 SEQ ID NO: 37 ctctgagcaaaatatccag 1121 1140 SEQ ID NO: 1040 ctggttactgagctgagag 1161 1180 1 6 SEQ ID NO: 38 atgaagcagtcacatctct 1189 1208 SEQ ID NO: 1041 agagctgccagtccttcat 10016 10035 1 6 SEQ ID NO: 39 ttgccacagctgattgagg 1209 1228 SEQ ID NO: 1042 cctcctacagtggtggcaa 4222 4241 1 6 SEQ ID NO: 40 agctgattgaggtgtccag 1216 1235 SEQ ID NO: 1043 ctggattccacatgcagct 11847 11866 1 6 SEQ ID NO: 41 tgctccactcacatcctcc 1278 1297 SEQ ID NO: 1044 ggaggctttaagttcagca 7601 7620 1 6 SEQ ID NO: 42 tgaaacgtgtgcatgccaa 1303 1322 SEQ ID NO: 1045 ttgggagagacaagtttca 8500 6519 1 6 SEQ ID NO: 43 gacattgctaattacctga 1503 1522 SEQ ID NO: 1046 tcagaagctaagcaatgtc 7232 7251 1 6 SEQ ID NO: 44 ttcttcttcagactttcct 1738 1757 SEQ ID NO: 1047 aggagagtccaaattagaa 8498 8517 1 6 SEQ ID NO: 45 ccaatatcttgaactcaga 1903 1922 SEQ ID NO: 1048 tctgaattcattcaattgg 6485 6504 1 6 SEQ ID NO: 46 aaagttagtgaaagaagtt 1946 1965 SEQ ID NO: 1049 aactaccctcactgccttt 2192 2151 1 6 SEQ ID NO: 47 aagttagtgaaagaagttc 1947 1966 SEQ ID NO: 1050 gaacctctggcatttactt 5916 5935 1 6 SEQ ID NO: 48 aaagaagttctgaaagaat 1956 1975 SEQ ID NO: 1051 attctctggtaactacttt 5482 5501 1 6 SEQ ID NO: 49 tttggctataccaaagatg 2322 2341 SEQ ID NO: 1052 catcttaggcactgacaaa 4997 5016 1 6 SEQ ID NO: 50 tgttgagaagctgattaaa 2381 2400 SEQ ID NO: 1053 tttagccatcggctcaaca 5700 5719 1 6 SEQ ID NO: 51 caggaagggctcaaagaat 2581 2580 SEQ ID NO: 1054 attcctttaacaattcctg 9492 9511 1 6 SEQ ID NO: 52 aggcagggctcaaagaatg 2562 2581 SEQ ID NO: 1055 cattcctttaaccattcct 9491 9510 1 6 SEQ ID NO: 53 gaagggctcaaagaatgac 2564 2583 SEQ ID NO: 1056 gtcagtcttcaggctcttc 7914 7933 1 6 SEQ ID NO: 54 caaagaatgacttttttct 2572 2591 SEQ ID NO: 1057 agaagatggcatttttttg 14000 14019 1 6 SEQ ID NO: 55 catggagaatgcctttgaa 2603 2822 SEQ ID NO: 1058 ttcagagccaaagtccatg 7119 7138 1 6 SEQ ID NO: 56 ggagccaaggctggagtaa 2879 2698 SEQ ID NO: 1059 ttactccaacgccagctcc 3050 3069 1 6 SEQ ID NO: 57 tcattccttccccaaagag 2884 2903 SEQ ID NO: 1060 ctctctggggcatctatga 5139 5158 1 6 SEQ ID NO: 58 acctatgagctccagagag 3165 3184 SEQ ID NO: 1061 ctctcaagaccacagaggt 12976 12995 1 6 SEQ ID NO: 59 gggcaaaacgtcttacaga 3365 3384 SEQ ID NO: 1062 tctgaaagacaacgtgccc 12317 12336 1 6 SEQ ID NO: 60 accctggacattcagaaca 3387 3406 SEQ ID NO: 1063 tgttgctaaggttcagggt 5675 5694 1 6 SEQ ID NO: 61 atgggcgacctaagttgtg 3429 3448 SEQ ID NO: 1064 cacaaattagtttcaccat 8941 8960 1 6 SEQ ID NO: 62 gatgaagagaagattgaat 3618 3837 SEQ ID NO: 1065 attccagcttccccacatc 8330 8349 1 6 SEQ ID NO: 63 caatgtagataccaaaaaa 3656 3875 SEQ ID NO: 1066 ttttttggaaatgccattg 8643 8682 1 6 SEQ ID NO: 64 gtagataccaaaaaaatga 3660 3679 SEQ ID NO: 1067 tcatgtgatgggtctctac 4371 4390 1 6 SEQ ID NO: 65 gcttcagttcatttggact 4509 4528 SEQ ID NO: 1068 agtcaagaaggacttaagc 5304 5323 1 6 SEQ ID NO: 66 tttgtttgtcaaagaagtc 4544 4563 SEQ ID NO: 1069 gacttcagagaaatacaaa 11400 11419 1 6 SEQ ID NO: 67 ttgtttgtcaaagaagtca 4545 4564 SEQ ID NO: 1070 tgacttcagagaaatacaa 11389 11418 1 6 SEQ ID NO: 68 tggcaatgggaaactcgct 5848 5865 SEQ ID NO: 1071 agcgagaatcaccctacca 8219 8238 1 6 SEQ ID NO: 69 aacctctggcatttacttt 5917 5936 SEQ ID NO: 1072 aaaggagatgtcaagggtt 10599 10818 1 6 SEQ ID NO: 70 catttactttctctcatga 5928 5945 SEQ ID NO: 1073 tcatttgaaagaataaatg 7026 7045 1 6 SEQ ID NO: 71 aaagtcagtgccctgctta 6009 6028 SEQ ID NO: 1074 taagaaccttactgacttt 7784 7803 1 6 SEQ ID NO: 72 tcccattttttgagacctt 6322 6341 SEQ ID NO: 1075 aaggacttcaggaatggga 12004 12023 1 6 SEQ ID NO: 73 catcaatattgatcaattt 6413 6432 SEQ ID NO: 1076 aaattaaaaagtcttgatg 6732 6751 1 6 SEQ ID NO: 74 taaagatagttatgattta 6665 6684 SEQ ID NO: 1077 taaaccaaaacttggttta 9019 9038 1 6 SEQ ID NO: 75 tattgatgaaatcattgaa 6713 6732 SEQ ID NO: 1078 ttcaaagacttaaaaaata 8007 8026 1 6 SEQ ID NO: 76 atgatctacatttgtttat 6790 6809 SEQ ID NO: 1079 ataaagaaattaaagtcat 7380 7399 1 6 SEQ ID NO: 77 agagacacatacagaatat 8919 6938 SEQ ID NO: 1080 atatattgtcagtgcctct 13382 13401 1 6 SEQ ID NO: 78 gacacatacagaatataga 8922 6941 SEQ ID NO: 1081 tctaaattcagttcttgtc 11327 11346 1 6 SEQ ID NO: 79 agcatgtcaaacactttgt 7054 7073 SEQ ID NO: 1082 acaaagtcagtgccctgct 6007 6028 1 6 SEQ ID NO: 80 tttttagaggaaaccaagg 7515 7534 SEQ ID NO: 1083 cctttgtgtacaccaaaaa 11230 11249 1 6 SEQ ID NO: 81 ttttagaggaaaccaaggc 7516 7535 SEQ ID NO: 1084 gcctttgtgtacaccaaaa 11229 11248 1 6 SEC ID NO: 82 ggaagatagacttcctgaa 9307 9326 SEQ ID NO: 1085 ttcagaaatactgttttcc 12824 12843 1 6 SEQ ID NO: 83 cactgtttctgagtcccag 9334 9353 SEQ ID NO: 1086 ctgggacctaccaagagtg 12523 12542 1 6 SEQ ID NO: 84 cacaaatcctttggctgtg 9888 9687 SEQ ID NO: 1087 cacatttcaaggaattgtg 10063 10082 1 6 SEQ ID NO: 85 ttcctggatacactgttcc 9853 9872 SEQ ID NO: 1088 ggaactgttgactcaggaa 12569 12588 1 6 SEQ ID NO: 86 gaaatctcaagctttctct 10042 10061 SEQ ID NO: 1089 agagccaggtcgagctttc 11044 11063 1 6 SEQ ID NO: 87 tttcttcatcttcatctgt 10210 10229 SEQ ID NO: 1090 acagctgaaagagatgaaa 13055 13074 1 6 SEQ ID NO: 88 tctaccgctaaaggagcag 10521 10540 SEQ ID NO: 1091 ctacacgctttgaggtaga 11781 11780 1 6 SEQ ID NO: 89 ctaccgctaaaggagcagt 10522 10541 SEQ ID NO: 1092 actgcacgctttgaggtag 11760 11779 1 6 SEQ ID NO: 90 agggcctctttttcaccaa 10831 10850 SEQ ID NO: 1093 ttggccaggaagtggccct 10957 10978 1 6 SEQ ID NO: 91 ttctccatccctgtaaaag 11265 11284 SEQ ID NO: 1094 ctttttcaccaacggagaa 10838 10857 1 6 SEQ ID NO: 92 gaaaaacaaagcagattat 11816 11835 SEQ ID NO: 1095 ataaactgcaagatttttc 13800 13619 1 6 SEQ ID NO: 93 actcactcattgattttct 12682 12701 SEQ ID NO: 1096 agaaaatcaggatctgagt 14027 14046 1 6 SEQ ID NO: 94 taaactaatagatgtaatc 12890 12909 SEQ ID NO: 1097 gattaccaccagcagttta 13578 13597 1 6 SEQ ID NO: 95 caaaacgagcttcaggaag 13200 13219 SEQ ID NO: 1098 cttcgtgaagaatattttg 13260 13279 1 6 SEQ ID NO: 96 tggaataatgctcagtgtt 2366 2385 SEQ ID NO: 1099 aacacttacttgaattcca 10662 10681 3 5 SEQ ID NO: 97 gatttgaaatccaaagaag 2400 2419 SEQ ID NO: 1100 cttcagagaaatacaaatc 11402 11421 3 5 SEQ ID NO: 98 atttgaaatccaaagaagt 2401 2420 SEQ ID NO: 1101 acttcagagaaatacaaat 11401 11420 3 5 SEQ ID NO: 99 atcaacagccgcttctttg 990 1009 SEQ ID NO: 1102 caaagaagtcaagattgat 4553 4572 2 5 SEQ ID NO: 100 tgttttgaagactctccag 1082 1101 SEQ ID NO: 1103 ctggaaagttaaaacaaca 6955 6974 2 5 SEQ ID NO: 101 cccttctgatagatgtggt 1324 1343 SEQ ID NO: 1104 accaaagctggcaccaggg 13961 13980 2 5 SEQ ID NO: 102 tgagcaagtgaagaacttt 1868 1887 SEQ ID NO: 1105 aaagccattcagtctctca 12963 12982 2 5 SEQ ID NO: 103 atttgaaatccaaagaagt 2401 2420 SEQ ID NO: 1106 acttttctaaacttgaaat 9055 9074 2 5 SEQ ID NO: 104 atccaaagaagtcccggaa 2408 2427 SEQ ID NO: 1107 ttccggggacacctgggat 12721 12740 2 5 SEQ ID NO: 105 agagcctacctccgcatct 2430 2449 SEQ ID NO: 1108 agatggtacgttagcctct 11921 11940 2 5 SEQ ID NO: 106 aatgcctttgaactcccca 2810 2629 SEQ ID NO: 1109 tgggaactacaatttcatt 7012 7031 2 5 SEQ ID NO: 107 gaagtcccaattccggatt 3297 3316 SEQ ID NO: 1110 aatcttcaatttattcttc 13815 13634 2 5 SEQ ID NO: 108 tgcaagcagaagccagaag 3496 3515 SEQ ID NO: 1111 cttcaggttccatcgtgca 11376 11395 2 5 SEQ ID NO: 109 gaagagcagattgaatttg 3621 3640 SEQ ID NO: 1112 caaaacctactatctcttc 10459 10478 2 5 SEQ ID NO: 110 atgctaaaggcacctatgg 4597 4616 SEQ ID NO: 1113 ccatatgaaagtcaagcat 12658 12875 2 5 SEQ ID NO: 111 tccctcacctccacctctg 4737 4756 SEQ ID NO: 1114 cagattctcagatgaggga 8912 8931 2 5 SEQ ID NO: 112 atttacagctctgacaagt 5427 5446 SEQ ID NO: 1115 acttttctaaacttgaaat 9055 9074 2 5 SEQ ID NO: 113 aggagcctaccaaaataat 5594 5613 SEQ ID NO: 1116 attatgttgaaacagtcct 11830 11849 2 5 SEQ ID NO: 114 aaagctgaagcacatcaat 6401 6420 SEQ ID NO: 1117 attgttgctcatctccttt 10194 10213 2 5 SEQ ID NO: 115 ctgctggaaacaacgagaa 9418 9437 SEQ ID NO: 1118 ttctgattaccaccagcag 13574 13593 2 5 SEQ ID NO: 116 ttgaaggaattcttgaaaa 9582 9801 SEQ ID NO: 1119 ttttaaaagaaatcttcaa 13805 13824 2 5 SEQ ID NO: 117 gaagtaaaagaaaattttg 10743 10762 SEQ ID NO: 1120 caaaacctactgtctcttc 10459 10478 2 5 SEQ ID NO: 118 tgcagaagatggcaaattt 11984 12003 SEQ ID NO: 1121 aaatgtccgctcttgttca 10894 10913 2 5 SEQ ID NO: 119 aggatctgagttattttgc 14035 14054 SEQ ID NO: 1122 gcaagtcagcccagttcct 10920 10939 2 5 SEQ ID NO: 120 gtgcccttctcggttgctg 18 37 SEQ ID NO: 1123 cagccattgacatgagcac 6740 5759 1 5 SEQ ID NO: 121 ggcgctacctgcgctgctg 148 165 SEQ ID NO: 1124 cagctccacagactccgcc 3062 3081 1 5 SEQ ID NO: 122 ctgcgctgctgctgctgct 154 173 SEQ ID NO: 1125 agcagaaggtgcgaagcag 3224 3243 1 5 SEQ ID NO: 123 gctgctggcgggcgccagg 170 189 SEQ ID NO: 1126 cctggattccacatgcagc 11848 11865 1 5 SEQ ID NO: 124 aagaggaaatgctggaaaa 193 212 SEQ ID NO: 1127 tttttcttcactacatctt 2584 2803 1 5 SEQ ID NO: 125 ctggaaaatgtcagcctgg 204 223 SEQ ID NO: 1128 ccagacttccacatcccag 3915 3934 1 5 SEQ ID NO: 126 tggagtccctgggactgct 296 315 SEQ ID NO: 1129 agcatgcctagtttctcca 9945 9964 1 5 SEQ ID NO: 127 ggagtccctgggcctgctg 297 318 SEQ ID NO: 1130 cagcatgcctagtttctcc 9944 9963 1 5 SEQ ID NO: 128 tgggactgctgattcaaga 305 324 SEQ ID NO: 1131 tcttccatcacttgaccca 2042 2081 1 5 SEQ ID NO: 129 ctgctgattcaagaagtgc 310 329 SEQ ID NO: 1132 gcacaccttgacattgcag 11079 11098 1 5 SEQ ID NO: 130 tgccaccaggatcaactgc 326 345 SEQ ID NO: 1133 gcaggctgaactggtggca 2717 2738 1 5 SEQ ID NO: 131 gccaccaggatcaactgca 327 348 SEQ ID NO: 1134 tgcaggctgaactggtggc 2716 2735 1 5 SEQ ID NO: 132 tgcaaggttgagctggagg 342 361 SEQ ID NO: 1135 cttgaacacaaagtcagtg 4744 4783 1 5 SEQ ID NO: 133 caaggttgagctggaggtt 344 363 SEQ ID NO: 1136 aacccctacatgaagcttg 13755 13774 1 5 SEQ ID NO: 134 ctctgcagcttcatcctga 369 388 SEQ ID NO: 1137 tcaggaagcttctcaagag 13211 13230 1 5 SEQ ID NO: 135 cagcttcatcctgaagacc 374 393 SEQ ID NO: 1138 ggtcttgagttaaatgctg 4977 4998 1 5 SEQ ID NO: 136 gcttcatcctgaagaccag 376 395 SEQ ID NO: 1139 ctggacgctaagaggaagc 855 874 1 5 SEQ ID NO: 137 tcatcctgaagaccagcca 379 398 SEQ ID NO: 1140 tggcatggcattatgatga 3604 3623 1 5 SEQ ID NO: 138 gaaaaccaagaactctgag 452 471 SEQ ID NO: 1141 ctcaaccttaatgattttc 8286 8305 1 5 SEQ ID NO: 139 agaactctgaggagtttgc 480 479 SEQ ID NO: 1142 gcaagctatacagtattct 8377 8398 1 5 SEQ ID NO: 140 tctgaggagtttgctgcag 465 484 SEQ ID NO: 1143 ctgcaggggatcccccagc 2526 2548 1 5 SEQ ID NO: 141 tttactgcagccatgtcca 474 493 SEQ ID NO: 1144 tggaagtgtcagtggcaaa 10372 10391 1 5 SEQ ID NO: 142 caagaggggcatcatttct 578 597 SEQ ID NO: 1145 agaataaatgacgttcttg 7035 7054 1 5 SEQ ID NO: 143 tcactttaccgtcaagacg 674 693 SEQ ID NO: 1146 cgtctacactatcatgtga 4380 4379 1 5 SEQ ID NO: 144 tttaccgtcaagacgagga 678 697 SEQ ID NO: 1147 tccttgacatgttgataaa 7366 7385 1 5 SEQ ID NO: 145 cactggacgctaagaggaa 853 872 SEQ ID NO: 1148 ttccagaaagcagccagtg 12498 12517 1 5 SEQ ID NO: 146 aggaagcatgtggcagaag 867 886 SEQ ID NO: 1149 cttcatacacattaatcct 9988 10007 1 5 SEQ ID NO: 147 caaggagcaacacctcttc 893 912 SEQ ID NO: 1150 gaagtagtactgcatcttg 6835 8854 1 5 SEQ ID NO: 148 acagactttgaaacttgaa 959 978 SEQ ID NO: 1151 ttcaattcttcaatgctgt 10500 10519 1 5 SEQ ID NO: 149 tgatgaagcagtcacatct 1187 1208 SEQ ID NO: 1152 agatttgaggattccatca 7978 7995 1 5 SEQ ID NO: 150 agcagtcacatc1ctcttg 1193 1212 SEQ ID NO: 1153 caaggagaaactgactgct 6524 6543 1 5 SEQ ID NO: 151 ccagccccattactttaca 1231 1250 SEQ ID NO: 1154 tgtagtctcctggtgctgg 5094 5113 1 5 SEQ ID NO: 152 ctccactcacatcctccag 1280 1299 SEQ ID NO: 1155 ctggagcttagtaatggag 8709 8728 1 5 SEQ ID NO: 153 catgccaacccccttctga 1314 1333 SEQ ID NO: 1156 tcagatgagggaacacatg 8919 8938 1 5 SEQ ID NO: 154 gagagatcttcaacatggc 1390 1409 SEQ ID NO: 1157 gccaccctggaactctctc 10889 10888 1 5 SEQ ID NO: 155 tcaacatggcgagggatca 1399 1418 SEQ ID NO: 1158 tgatcccacctctcattga 2985 2984 1 5 SEQ ID NO: 156 ccaccttgtatgcgctgag 1429 1448 SEQ ID NO: 1159 ctcagggatctgaaggtgg 8187 8208 1 5 SEQ ID NO: 157 gtcaacaactatcataaga 1455 1474 SEQ ID NO: 1160 tcttgagttaaatgctgac 4979 4998 1 5 SEQ ID NO: 158 tggacattgctaattacct 1501 1520 SEQ ID NO: 1161 aggtatattcgaaagtcca 12799 12818 1 5 SEQ ID NO: 159 ggacattgctaattacctg 1502 1521 SEQ ID NO: 1162 caggtatattcgaaagtcc 12798 12817 1 5 SEQ ID NO: 160 ttctgcgggtcattggaaa 1573 1592 SEQ ID NO: 1163 tttcacatgccaaggagaa 8514 8533 1 5 SEQ ID NO: 161 ccagaactcaagtcttcaa 1820 1639 SEQ ID NO: 1164 ttgaagtgtagtctcctgg 5088 5107 1 5 SEQ ID NO: 162 agtcttcaatcctgaactg 1630 1649 SEQ ID NO: 1165 catttctgattggtggact 7757 7778 1 5 SEQ ID NO: 163 tgagcaagtgaagaacttt 1868 1887 SEQ ID NO: 1166 aaagtgccacttttactca 6183 6202 1 5 SEQ ID NO: 164 agcaagtgaagaactttgt 1870 1889 SEQ ID NO: 1167 acaaagtcagtgccctgct 6007 6028 1 5 SEQ ID NO: 165 tctgaaagaatctcaactt 1964 1983 SEQ ID NO: 1168 aagtccataatggttcaga 12811 12830 1 5 SEQ ID NO: 166 actgtcatggacttcagaa 1986 2005 SEQ ID NO: 1169 ttctgaatatattgtcagt 13376 13395 1 5 SEQ ID NO: 167 acttgacccagcctcagcc 2051 2070 SEQ ID NO: 1170 ggctcaccctgagagaagt 12391 12410 1 5 SEQ ID NO: 168 tccaaataactaccttcct 2096 2115 SEQ ID NO: 1171 aggaagatatgaagatgga 4712 4731 1 5 SEQ ID NO: 169 actaccctcactgcctttg 2133 2152 SEQ ID NO: 1172 caaatttgtggagggtagt 10319 10338 1 5 SEQ ID NO: 170 ttggatttgcttcagctga 2149 2168 SEQ ID NO: 1173 tcagtataagtacaaccaa 9392 9411 1 5 SEQ ID NO: 171 ttggssgctctttttggga 2211 2230 SEQ ID NO: 1174 tcccgattcacgcttccaa 11577 11596 1 5 SEQ ID NO: 172 ggaagctctttttgggaag 2213 2232 SEQ ID NO: 1175 cttcagaaagctaccttcc 7929 7948 1 5 SEQ ID NO: 173 tttttcccagacagtgtca 2238 2257 SEQ ID NO: 1176 tgaccttctctaagcaaaa 4878 4895 1 5 SEQ ID NO: 174 agacagtgtcaacaaagct 2248 2285 SEQ ID NO: 1177 agcttggttttgccagtct 2458 2477 1 5 SEQ ID NO: 175 ctttggctataccaaagat 2321 2340 SEQ ID NO: 1178 atctcgtgtctaggaaaag 5968 6987 1 5 SEQ ID NO: 176 caaagatgataaacatgag 2333 2352 SEQ ID NO: 1179 ctcaaggataacgtgtttg 12809 12628 1 5 SEQ ID NO: 177 gatatggtaaatggaataa 2355 2374 SEQ ID NO: 1180 ttatcttattaattatatc 13079 13098 1 5 SEQ ID NO: 178 ggcataatgctcagtgttg 2367 2386 SEQ ID NO: 1181 caacacttacttgaattcc 10881 10880 1 5 SEQ ID NO: 179 tttgaaatccaaagaagtc 2402 2421 SEQ ID NO: 1182 gacttcagagaaatacaaa 11400 11419 1 5 SEQ ID NO: 180 gatcccccagatgattgga 2534 2553 SEQ ID NO: 1183 tccaatttccctgtggatc 3681 3700 1 5 SEQ ID NO: 181 cagatgattggagaggtca 2541 2560 SEQ ID NO: 1184 tgaccacacaaacagtctg 5363 5382 1 5 SEQ ID NO: 182 agaatgacttttttcttca 2575 2594 SEQ ID NO: 1185 tgaagtccggattcattct 11015 11034 1 5 SEQ ID NO: 183 gaactccccactggagctg 2619 2638 SEQ ID NO: 1186 cagctcaaccgtacagttc 11861 11880 1 5 SEQ ID NO: 184 atatcttcatctggagtca 2652 2671 SEQ ID NO: 1187 tgacttcagtgcagaatat 11966 11985 1 5 SEQ ID NO: 185 gtcattgctcccggagcca 2667 2688 SEQ ID NO: 1188 tggccccgtttaccatgac 5809 5828 1 5 SEQ ID NO: 186 gctgaagtttatcattcct 2873 2892 SEQ ID NO: 1189 aggaggctttaagttcagc 7600 7619 1 5 SEQ ID NO: 187 attccttccccaaagagac 2886 2905 SEQ ID NO: 1190 gtctcttcctccatggaat 10470 10489 1 5 SEQ ID NO: 188 ctcattgagaacaggcagt 2976 2995 SEQ ID NO: 1191 actgactgcacgctttgag 11756 11775 1 5 SEQ ID NO: 189 ttgagcagtattctctcag 3142 3161 SEQ ID NO: 1192 ctgagagaagtgtcttcaa 12399 12418 1 5 SEQ ID NO: 190 accttgtccagtgaagtcc 3285 3304 SEQ ID NO: 1193 ggacggtactgtcccaggt 12784 12803 1 5 SEQ ID NO: 191 ccagtgaagtccaaattcc 3292 3311 SEQ ID NO: 1194 ggaaggcagagtttactgg 9148 9167 1 5 SEQ ID NO: 182 acattcagaacaagaaaat 3394 3413 SEQ ID NO: 1195 atttcctaaagctggatgt 11167 11186 1 5 SEQ ID NO: 193 gaaaaatcaagggtgttat 3483 3482 SEQ ID NO: 1196 ataaactgcaagatttttc 13600 13619 1 5 SEQ ID NO: 194 aaatcaagggtgttatttc 3466 3485 SEQ ID NO: 1197 gaaacaatgcattagattt 9745 9764 1 5 SEQ ID NO: 195 tggcattatgatgaagaga 3609 3628 SEQ ID NO: 1198 tctcccgtgtataatgcca 11781 11800 1 5 SEQ ID NO: 196 aagagaagattgaatttga 3622 3641 SEQ ID NO: 1199 tcaaaacctactgtctctt 10458 10477 1 5 SEQ ID NO: 197 aaatgacttccaatttccc 3673 3692 SEQ ID NO: 1200 gggaactacaatttcattt 7013 7032 1 5 SEQ ID NO: 198 atgacttccaatttccctg 3675 3694 SEQ ID NO: 1201 caggctgattacgagtcat 4917 4936 1 5 SEQ ID NO: 199 acttccaatttccctgtgg 3678 3697 SEQ ID NO: 1202 ccacgaaaaatatggaagt 10380 10379 1 5 SEQ ID NO: 200 agttgcaatgagctcatgg 3803 3822 SEQ ID NO: 1203 ccatcagttcagataaact 7989 8008 1 5 SEQ ID NO: 201 tttgcaagaccacctcaat 3860 3879 SEQ ID NO: 1204 attgacctgtccattcaaa 13671 13690 1 5 SEQ ID NO: 202 gaaggagacaacctccagc 3884 3903 SEQ ID NO: 1205 ctggaattgtcattccttc 11728 11747 1 5 SEQ ID NO: 203 acttccacatcccagaaaa 3919 3938 SEQ ID NO: 1206 ttttaacaaaagtggaagt 6821 6840 1 5 SEQ ID NO: 204 ctcttcttaaaaagcgatg 3939 3958 SEQ ID NO: 1207 catcactgccaaaggagag 8486 8505 1 5 SEQ ID NO: 205 cacagcgatggcccggtca 3948 3967 SEQ ID NO: 1208 tgactcactcattgatttt 12880 12699 1 5 SEQ ID NO: 206 ttcdttgccattggtggca 4003 4022 SEQ ID NO: 1209 ccacaaacaatgaagggaa 9258 9275 1 5 SEQ ID NO: 207 caagtctgtgggattccat 4079 4098 SEQ ID NO: 1210 atgggaaaaaacaggcttg 9566 9585 1 5 SEQ ID NO: 208 aagtccctacttttaccat 4117 4136 SEQ ID NO: 1211 atgggaagtataagaactt 4834 4853 1 5 SEQ ID NO: 209 tgcctctcctgggtgttct 4159 4178 SEQ ID NO: 1212 agaaaaacaaacacaggca 9843 9662 1 5 SEQ ID NO: 210 accagcacagaccatttca 4242 4261 SEQ ID NO: 1213 tgaagtgtagtctcctggt 5089 5108 1 5 SEQ ID NO: 211 ccagcacagaccatttcag 4243 4262 SEQ ID NO: 1214 ctgaaatacaatgctctgg 5511 5530 1 5 SEQ ID NO: 212 actatcatgtgatgggtct 4367 4386 SEQ ID NO: 1215 agacacctgattttatagt 7948 7967 1 5 SEQ ID NO: 213 accacagatgtctgcttca 4496 4515 SEQ ID NO: 1216 tgaaggctgactctgtggt 4282 4301 1 5 SEQ ID NO: 214 ccacagatgtctgcttcag 4497 4516 SEQ ID NO: 1217 ctgagcaacaaatttgtgg 10311 10330 1 5 SEQ ID NO: 215 tttggactccaaaaagaaa 4520 4539 SEQ ID NO: 1218 tttctctcatgattacaaa 5933 5952 1 5 SEQ ID NO: 216 tcaaagaagtcaagattga 4552 4571 SEQ ID NO: 1219 tcaaggataacgtgtttga 12610 12629 1 5 SEQ ID NO: 217 atgagaactacgagctgac 4798 4817 SEQ ID NO: 1220 gtcagatattgttgctcat 10187 10206 1 5 SEQ ID NO: 218 ttaaaatctgacaccaatg 4818 4837 SEQ ID NO: 1221 cattcattgaagatgttaa 7342 7361 1 5 SEQ ID NO: 219 gaagtataagaactttgcc 4838 4857 SEQ ID NO: 1222 ggcaaatttgaaggacttc 11994 12013 1 5 SEQ ID NO: 220 aagtataagaactttgcca 4839 4858 SEQ ID NO: 1223 tggcaaatttgaaggactt 11993 12012 1 5 SEQ ID NO: 221 ttcttcagcctgctttctg 4941 4960 SEQ ID NO: 1224 cagaatccagatacaagaa 6884 6903 1 5 SEQ ID NO: 222 ctggatcactaaattccca 4957 4976 SEQ ID NO: 1225 tggatctttccagagccag 11033 11052 1 5 SEQ ID NO: 223 aaattaatagtggtgctca 5014 5033 SEQ ID NO: 1226 tgagaagccccaagaattt 6248 6267 1 5 SEQ ID NO: 224 agtgcaacgaccaacttga 5073 5092 SEQ ID NO: 1227 tcaaattcctggatacact 9848 9867 1 5 SEQ ID NO: 225 ctgggaagtgcttatcagg 5238 5257 SEQ ID NO: 1228 cctgaccttcacataccag 8310 8329 1 5 SEQ ID NO: 226 gcaaaaacattttcaactt 5278 5297 SEQ ID NO: 1229 aagtaaaagaaaattttgc 10744 10763 1 5 SEQ ID NO: 227 aaaaacattttcaacttca 5280 5299 SEQ ID NO: 1230 tgaagtaaaagaaaatttt 10742 10761 1 5 SEQ ID NO: 228 tcagtcaagaaggacttaa 5302 5321 SEQ ID NO: 1231 ttaaggacttccattctga 13363 13382 1 5 SEQ ID NO: 229 tcaaatgacatgatgggct 5325 5344 SEQ ID NO: 1232 agcccatcaatatcattga 6205 6224 1 5 SEQ ID NO: 230 cacacaaacagtctgaaca 5367 5386 SEQ ID NO: 1233 tgtttcaactgcctttgtg 11219 11238 1 5 SEQ ID NO: 231 tcttcaaaacttgacaaca 5409 5428 SEQ ID NO: 1234 tgttttcctatttccaaga 12835 12864 1 5 SEQ ID NO: 232 caagttttataagcaaact 5441 5460 SEQ ID NO: 1235 agttattttgctaaacttg 14043 14062 1 5 SEQ ID NO: 233 tggtaactactttaaacag 5488 5507 SEQ ID NO: 1236 ctgtttttagaggaaacca 7512 7531 1 5 SEQ ID NO: 234 aacagtgacctgaaataca 5502 5521 SEQ ID NO: 1237 tgtatagcaaattcctgtt 5890 5909 1 5 SEQ ID NO: 235 gggaaactacggctagaac 5544 5563 SEQ ID NO: 1238 gttccttccatgatttccc 10933 10952 1 5 SEQ ID NO: 236 aacacatctatgccatctc 5620 5639 SEQ ID NO: 1239 gagacagcatcttcgtgtt 11204 11223 1 5 SEQ ID NO: 237 tcagcaagctataaagcag 5652 5671 SEQ ID NO: 1240 ctgctaagaaccttactga 7780 7799 1 5 SEQ ID NO: 238 gcagccactgttgctaagg 5667 5686 SEQ ID NO: 1241 cctttcaagcactgactgc 11746 11765 1 5 SEQ ID NO: 239 tctggggagaacatactgg 5866 5885 SEQ ID NO: 1242 ccaggttttccacaccaga 8038 8057 1 5 SEQ ID NO: 240 ttctctcatgattacaaag 5934 5953 SEQ ID NO: 1243 ctttttcaccaacggagaa 10838 10857 1 5 SEQ ID NO: 241 ctgagcagacaggcacctg 6034 6053 SEQ ID NO: 1244 caggaggctttaagttcag 7599 7618 1 5 SEQ ID NO: 242 caatttaacaaccatgaat 6066 6085 SEQ ID NO: 1245 attccttcctttacaattg 8082 8101 1 5 SEQ ID NO: 243 tggacgaactctggctgac 6140 6159 SEQ ID NO: 1246 gtcagcccagttccttcca 10924 10943 1 5 SEQ ID NO: 244 cttttactcagtgagccca 6192 6211 SEQ ID NO: 1247 tgggctaaacgtatgaaag 7827 7846 1 5 SEQ ID NO: 245 tcattgatgctttagagat 6217 6238 SEQ ID NO: 1248 atcttcataagttcaatga 13174 13193 1 5 SEQ ID NO: 246 aaaaccaagatgttcactc 6295 6314 SEQ ID NO: 1249 gagtgaaatgctgtttttt 8630 8649 1 5 SEQ ID NO: 247 aggaatcgacaaaccatta 6357 6378 SEQ ID NO: 1250 taatgattttcaagttcct 8294 8313 1 5 SEQ ID NO: 248 tagttgtactggaaaacgt 6376 6395 SEQ ID NO: 1251 acgttagcctctaagacta 11928 11947 1 5 SEQ ID NO: 249 ggaaaacgtacagagaaag 6388 6405 SEQ ID NO: 1252 cttttacaattcattttcc 13014 13033 1 5 SEQ ID NO: 250 gaaaacgtacagagaaagc 6387 6406 SEQ ID NO: 1253 gctttctcttccacatttc 10052 10071 1 5 SEQ ID NO: 251 aaagctgaagcacatcaat 6401 6420 SEQ ID NO: 1254 attgatgttagagtgcttt 6984 7003 1 5 SEQ ID NO: 252 aagctgaagcacatcaata 6402 6421 SEQ ID NO: 1255 tattgatgttagagtgctt 6983 7002 1 5 SEQ ID NO: 253 tgaagcacatcaatattga 6406 6425 SEQ ID NO: 1256 tcaaccttaatgattttca 8287 8306 1 5 SEQ ID NO: 254 atcaatattgatcaatttg 6414 6433 SEQ ID NO: 1257 caaagccatcactgatgat 1680 1679 1 5 SEQ ID NO: 255 taatgattatctgaattca 6476 6495 SEQ ID NO: 1258 tgaaatcattgaaaaatta 6719 8738 1 5 SEQ ID NO: 256 gattatctgaattcattca 6480 6499 SEQ ID NO: 1259 tgaagtagctgagaaaatc 7094 7113 1 5 SEQ ID NO: 257 aattgggagagacaagttt 6498 6517 SEQ ID NO: 1260 aaacattcctttaacaatt 9488 9507 1 5 SEQ ID NO: 258 aaaatagctattgctaata 6593 6712 SEQ ID NO: 1261 tattgaaaatattgatttt 6806 8825 1 5 SEQ ID NO: 259 aaaattaaaaagtcttgat 6731 6750 SEQ ID NO: 1262 atcatatccgtgtaatttt 6757 6776 1 5 SEQ ID NO: 260 ttgaaaatattgattttaa 6808 6827 SEQ ID NO: 1263 ttaatcttcataagttcaa 13171 13190 1 5 SEQ ID NO: 261 agacatccagcacctagct 6938 6957 SEQ ID NO: 1264 agcttggttttgccagtct 2458 2477 1 5 SEQ ID NO: 262 caatttcatttgaaagaat 7021 7040 SEQ ID NO: 1265 attccttcctttacaattg 8082 8101 1 5 SEQ ID NO: 263 aggttttaatggataaatt 7174 7193 SEQ ID NO: 1266 aattgttgaaagaaaacct 13147 13188 1 5 SEQ ID NO: 264 cagaagctaagcaatgtcc 7233 7252 SEQ ID NO: 1267 ggacaaggcccagaatctg 12545 12584 1 5 SEQ ID NO: 265 taagataaaagattacttt 7262 7281 SEQ ID NO: 1268 aaagaaaacctatgcctta 13155 13174 1 5 SEQ ID NO: 266 aaagattactttgagaaat 7269 7288 SEQ ID NO: 1269 atttcttaaacattccttt 9481 9500 1 5 SEQ ID NO: 267 gagaaattagttggattta 7281 7300 SEQ ID NO: 1270 tcaagccattcagtctctc 12982 12981 1 5 SEQ ID NO: 268 atttattgatgatgctgtc 7295 7314 SEQ ID NO: 1271 gacatgttgataaagaaat 7371 7390 1 5 SEQ ID NO: 269 gaattatcttttaaaacat 7326 7345 SEQ ID NO: 1272 atgtatcaaatggacattc 7877 7696 1 5 SEQ ID NO: 270 ttaccaccagtttgtagat 7403 7422 SEQ ID NO: 1273 atctggaaccttgaagtaa 10731 10750 1 5 SEQ ID NO: 271 ttgcagtgtatctggaaag 7540 7559 SEQ ID NO: 1274 cttttcacattagatgcaa 8412 8431 1 5 SEQ ID NO: 272 cattcagcaggaacttcaa 7691 7710 SEQ ID NO: 1275 ttgaaggccttcaggaatg 12001 12020 1 5 SEQ ID NO: 273 acacctgattttatcgtcc 7950 7969 SEQ ID NO: 1276 ggactcaaggataacgtgt 12606 12625 1 5 SEQ ID NO: 274 ggattccatcagttcagat 7984 8003 SEQ ID NO: 1277 atcttcaatgattatatcc 13116 13136 1 5 SEQ ID NO: 275 ttgtagacatgaaagtaaa 8104 8123 SEQ ID NO: 1278 tttatgattatgtcaacaa 12352 12371 1 5 SEQ ID NO: 276 ctgaacagtgagctgcagt 8148 8187 SEQ ID NO: 1279 actggacttctctagtcag 8801 8820 1 5 SEQ ID NO: 277 aatccaatctcctcttttc 8399 8418 SEQ ID NO: 1280 gaaaaatgaagtccggatt 11009 11028 1 5 SEQ ID NO: 278 attttgattttcaagcaaa 8524 8543 SEQ ID NO: 1281 tttgcaagttaaagaaact 14015 14034 1 5 SEQ ID NO: 279 ttttgattttcaagcaaat 8525 8544 SEQ ID NO: 1282 atttgatttaagtgtaaaa 9814 9833 1 5 SEQ ID NO: 280 tgattttcaagcacatgca 8528 8647 SEQ ID NO: 1283 tgcaagttaaagaaaatca 14017 14036 1 5 SEQ ID NO: 281 atgctgttttttggaaatg 8637 8858 SEQ ID NO: 1284 cattggtaggagacagcat 11195 11214 1 5 SEQ ID NO: 282 tgctgttttttggaaatgc 8638 8657 SEQ ID NO: 1285 gcattggtaggagacagca 11194 11213 1 5 SEQ ID NO: 283 caaaaaatacactagagct 8898 8717 SEQ ID NO: 1286 agctagagggcctcttttt 10825 10844 1 5 SEQ ID NO: 284 actggagcttagtaatgga 8708 8727 SEQ ID NO: 1287 tccactcacatcctccagt 1281 1300 1 5 SEQ ID NO: 285 cttctggaaaagggtcatg 8878 8897 SEQ ID NO: 1288 catgaacccctacatgaag 13751 13770 1 5 SEQ ID NO: 286 ggcaaagggtcatggaaat 8883 8902 SEQ ID NO: 1289 atttgaaagttcgttttcc 9274 9293 1 5 SEQ ID NO: 287 gggcctgccccagattctc 8902 8921 SEQ ID NO: 1290 gagaacattatggaggccc 9432 9451 1 5 SEQ ID NO: 288 ttctcagatgagggaacac 8916 8935 SEQ ID NO: 1291 gtgtcttcaaagctgagaa 12408 12427 1 5 SEQ ID NO: 289 gatgagggaacacatgaat 8922 8941 SEQ 1D NO: 1292 attccagcttccccacatc 8330 8349 1 5 SEQ ID NO: 290 ctttggactgtccaataag 8978 8997 SEQ ID NO: 1293 cttatgggatttcctaaag 11159 11178 1 5 SEQ ID NO: 291 gcatccacaaacaatgaag 9252 9271 SEQ ID NO: 1294 cttcatctgtcattgatgc 10219 10238 1 5 SEQ ID NO: 292 cacaaacaatgaagggaat 9257 9276 SEQ ID NO: 1295 attccctgaagttgatgtg 11480 11499 1 5 SEQ ID NO: 293 ccaacatttctctgctgga 9407 9426 SEQ ID NO: 1296 tccatcacaaatcctttgg 9663 9682 1 5 SEQ ID NO: 294 caaaatttctctgctggaa 9408 9427 SEQ ID NO: 1297 ttccatcacaaatcctttg 9862 9681 1 5 SEQ ID NO: 295 tctgctggaaacaacgaga 9417 9438 SEQ ID NO: 1298 tctcaagagttacagcaga 13221 13240 1 5 SEQ ID NO: 296 ctgctggaaacaacgagaa 9418 9437 SEQ ID NO: 1299 ttctcaagagttacagcag 13220 13239 1 5 SEQ ID NO: 297 agaacattatggcggccca 9433 9452 SEQ ID NO: 1300 tgggcctgccccagattct 8901 8920 1 5 SEQ ID NO: 298 agaagcaaatctggatttc 9487 9488 SEQ ID NO: 1301 gaaatcttcaatttattct 13813 13832 1 5 SEQ ID NO: 299 tttctctctatgggaaaaa 9657 9576 SEQ ID NO: 1302 tttttgcaagttaaagaaa 14013 14032 1 5 SEQ ID NO: 300 tcagagcatcaaatccttt 9704 9723 SEQ ID NO: 1303 aaagaaaatcaggatctga 14025 14044 1 5 SEQ ID NO: 301 cagaaacaatgcattagat 9743 9782 SEQ ID NO: 1304 atctatgccatctcttctg 5625 5844 1 5 SEQ ID NO: 302 tacacattaatcctgccat 9993 10012 SEQ ID NO: 1305 atggagtctttattgtgta 14081 14100 1 5 SEQ ID NO: 303 agtcagatattgttgctca 10188 10205 SEQ ID NO: 1306 tgagaactacgagctgact 4799 4818 1 5 SEQ ID NO: 304 ggagggtagtcataacagt 10328 10347 SEQ ID NO: 1307 actggtggccaaaccctcc 2726 2745 1 5 SEQ ID NO: 305 caaaagccgaaattccaat 10396 10415 SEQ ID NO: 1308 attgaagtacctacttttg 8358 8377 1 5 SEQ ID NO: 306 aaaagccgaaattccaatt 10397 10416 SEQ ID NO: 1309 aattgaagtacctactttt 8357 8376 1 5 SEQ ID NO: 307 ttcaagcaagaacttaatg 10428 10447 SEQ ID NO: 1310 cattatggcccttcgtgaa 13250 13289 1 5 SEQ ID NO: 308 cctcttacttttccattga 10570 10589 SEQ ID NO: 1311 tcaaaagaagcccaagagg 12939 12958 1 5 SEQ ID NO: 309 tgaggccaacacttacttg 10655 10674 SEQ ID NO: 1312 caagcatctgattgactca 12868 12687 1 5 SEQ ID NO: 310 cacttacttgaattccaag 10664 10683 SEQ ID NO: 1313 cttgaacacaaagtcagtg 8000 6019 1 5 SEQ ID NO: 311 gaagtaaaagaaaattttg 10743 10762 SEQ ID NO: 1314 caaaaacattttcaacttc 5279 5298 1 5 SEQ ID NO: 312 cctggaactctctccatgg 10874 10893 SEQ ID NO: 1315 ccatttacagatcttcagg 11384 11383 1 5 SEQ ID NO: 313 agctggatgtacccaccag 11176 11195 SEQ ID NO: 1316 ctggattccacatgcagct 11847 11866 1 5 SEQ ID NO: 314 aaaattccctgaagttgat 11477 11496 SEQ ID NO: 1317 atcatatccgtgtaatttt 6757 6778 1 5 SEQ ID NO: 315 cagatggcattgctgcttt 11605 11624 SEQ ID NO: 1318 aaagctgagaagaaatctg 12416 12435 1 5 SEQ ID NO: 316 agatggcattgctgctttg 11806 11625 SEQ ID NO: 1319 caaagctgagaagaaatct 12415 12434 1 5 SEQ ID NO: 317 tgttgaaacagtcctggat 11834 11853 SEQ ID NO: 1320 atcccagatgagatcaacc 13095 13114 1 5 SEQ ID NO: 318 catattcaaaactgagttg 12221 12240 SEQ ID NO: 1321 caactctctgattactatg 13823 13642 1 5 SEQ ID NO: 319 aaagatttatcaaaagaag 12930 12949 StO ID NO: 1322 cttcaatttattcttcttt 13818 13837 1 5 SEQ ID NO: 320 attttccaactaatagaag 13028 13045 SEQ ID Nc: 1323 cttcaaagacttaaaaaat 8006 8025 1 5 SEQ ID NO: 321 aattatatccaagatgaga 13089 13108 SEQ ID NO: 1324 tctcttcctccatggaatt 10471 10490 1 5 SEQ ID NO: 322 ttcaggaagcttctcaaga 13210 13229 SEQ ID NO: 1325 tcttcataagttcaatgaa 13175 13194 1 5 SEQ ID NO: 323 ttgagcaatttctgcacag 13429 13448 SEQ ID NO: 1326 cttttgaaagatttatcaa 12924 12943 1 5 SEQ ID NO: 324 ctgatatacatcacggagt 13704 13723 SEQ ID NO: 1327 actcaatggtgaaattcag 7457 7476 1 5 SEQ ID NO: 325 acatcacggagttactgaa 13711 13730 SEQ ID NO: 1328 ttcagaagctaagcaatgt 7231 7250 1 5 SEQ ID NO: 326 actgcctatattgataaaa 13874 13893 SEQ ID NO: 1329 ttttggcaagctatacagt 8372 8391 1 5 SEQ ID NO: 327 aggatggcattttttgcaa 14003 14022 SEQ ID NO: 1330 ttgcaagcaagtctttcct 3005 3024 1 5 SEQ ID NO: 328 ttttttgcaagttaaagaa 14012 14031 SEQ ID NO: 1331 ttctctctatgggaaaaaa 9558 9577 1 5 SEQ ID NO: 329 tccagaactcaagtcttca 1819 1838 SEQ ID NO: 1332 tgaaatgctgttttttgga 8033 8852 3 4 SEQ ID NO: 330 agttagtgaaagaagttct 1948 1967 SEQ ID NO: 1333 agaatctgtaccaggaact 12556 12575 3 4 SEQ ID NO: 331 atttacagctctgacaagt 5427 5448 SEQ ID NO: 1334 acttcagagaaatacaaat 11401 11420 3 4 SEQ ID NO: 332 gattatctgaattcattca 8480 6499 SEQ ID NO: 1335 tgaaaccaatgacaaaatc 7421 7440 3 4 SEQ ID NO: 333 gtgcccactcggttgctgt 18 37 SEQ ID NO: 1336 cagctgagcagacaggcac 6031 6050 2 4 SEQ ID NO: 334 attcaagcacctccggaag 245 264 SEQ ID NO: 1337 cttcataagttcaatgaat 13176 13195 2 4 SEQ ID NO: 335 gactgctgattcaagaagt 308 327 SEQ ID NO: 1338 acttcccaactctcaagtc 13407 13428 2 4 SEQ ID NO: 336 ttgctgcagccatgtccag 476 494 SEQ ID NO: 1339 ctgggcagctgtatagcaa 5881 5900 2 4 SEQ ID NO: 337 agaaagatgaacctactta 547 566 SEQ ID NO: 1340 taagtatgatttcaattct 10490 10509 2 4 SEQ ID NO: 338 tgaagactctccaggaact 1087 1106 SEQ ID NO: 1341 agttcaatgaatttattca 13183 13202 2 4 SEQ ID NO: 339 atctctcttgccacagctg 1202 1221 SEQ ID NO: 1342 cagcccagccatttgagat 9229 9248 2 4 SEQ ID NO: 340 tctctcttgccacagctga 1203 1222 SEQ ID NO: 1343 tcagcccagccatttgaga 9228 9247 2 4 SEQ ID NO: 341 tgaggtgtccagccccatc 1223 1242 SEQ ID NO: 1344 gatgggaaagccgccctca 5208 5227 2 4 SEQ ID NO: 342 ccagcactcaagtcttcaa 1820 1839 SEQ ID NO: 1345 ttgaaagcagaacctctgg 5907 5928 2 4 SEQ ID NO: 343 ctgaaaaagttagtgaaag 1941 1960 SEQ ID NO: 1346 ctttctcgggaatattcag 10623 10842 2 4 SEQ ID NO: 344 tttttcccagacagtgtca 2238 2257 SEQ ID NO: 1347 tgacaggcattttgaaaac 9722 9741 2 4 SEQ ID NO: 345 ttttcccagacagtgtcaa 2239 2258 SEQ ID NO: 1348 ttgacaggcattttgaaaa 9721 9740 2 4 SEQ ID NO: 346 cattcagacccagaaaatt 3395 3414 SEQ ID NO: 1349 aattccaattttgagaatg 10406 10425 2 4 SEQ ID NO: 347 tgacgagaagattgaattt 3620 3639 SEQ ID NO: 1350 aaatgtcagctcttgttca 10894 10913 2 4 SEQ ID NO: 348 tttgaatggaacacaggca 3636 3655 SEQ ID NO: 1351 tgccagtttgaaaaacaca 11807 11828 2 4 SEQ ID NO: 349 ttctagattcgaatatcaa 4399 4418 SEQ ID NO: 1352 ttgacatgttgataaagaa 7369 7388 2 4 SEQ ID NO: 350 gattcgaatatcaaattca 4404 4423 SEQ ID NO: 1353 tgaagtagaccaaccaatc 7154 7173 2 4 SEQ ID NO: 351 tgcaacgaccaccttgaag 5075 5094 SEQ ID NO: 1354 cttcaggttccatcgtgca 11376 11395 2 4 SEQ ID NO: 352 ttaagctctcaaatgacat 5317 5336 SEQ ID NO: 1355 atgttgataaagaaattaa 7374 7393 2 4 SEQ ID NO: 353 ccatttaacaacaatgaat 8088 6085 SEQ ID NO: 1356 attcacactgcctatattg 13888 13887 2 4 SEQ ID NO: 354 tgactacagccaggacttg 8080 6099 SEQ ID NO: 1357 caagagcacacggtcttca 10679 10898 2 4 SEQ ID NO: 355 catcaatattgatcaattt 8413 6432 SEQ ID NO: 1358 aaattccctgaagttgatg 11478 11497 2 4 SEQ ID NO: 356 ttgagcatgtcaaacactt 7051 7070 SEQ ID NO: 1359 aagtaagtgctaggttcaa 9373 9392 2 4 SEQ ID NO: 357 tgaaggagactattcagaa 7219 7238 SEQ ID NO: 1360 ttctgcacagaaatattcc 13438 13457 2 4 SEQ ID NO: 358 ttcaggctcttcagaaagc 7921 7940 SEQ ID NO: 1361 gcttgctaacctctctgaa 12304 12323 2 4 SEQ ID NO: 359 tccacaaattgaacatccc 8779 8798 SEQ ID NO: 1362 gggacctaccaagagtgga 12525 12544 2 4 SEQ ID NO: 360 tgaataccaatgcttaact 10159 10178 SEQ ID NO: 1363 agttcaatgaatttattca 13183 13202 2 4 SEQ ID NO: 361 taaactaatagatgtaatc 12890 12909 SEQ ID NO: 1364 gattactatgaaaaattta 13832 13651 2 4 SEQ ID NO: 362 ttgccctgtccattcaaaa 13672 13691 SEQ ID NO: 1365 ttttaaaagaaatcttcaa 13805 13824 2 4 SEQ ID NO: 363 gggctgagtgcccttctcg 11 30 SEQ ID NO: 1366 cgaggacaggccgcagccc 76 95 1 4 SEQ ID NO: 364 ggctgagtgcccttctcgg 12 31 SEQ ID NO: 1367 ccgaggccaggccgcagcc 75 94 1 4 SEQ ID NO: 365 ctgagtgccctcttcggtt 14 33 SEQ ID NO: 1368 aaccgtgcctgaatctcag 11549 11568 1 4 SEQ ID NO: 366 tctcggttgctgccgctga 25 44 SEQ ID NO: 1369 tcagctgacctcatcgaga 2180 2179 1 4 SEQ ID NO: 367 caggccgcagcccaggagc 82 101 SEQ ID NO: 1370 gctctgcagcttcatcctg 368 387 1 4 SEQ ID NO: 368 gctggcgctgcctgcgctg 143 182 SEQ ID NO: 1371 cagcacagaccatttcagc 4244 4283 1 4 SEQ ID NO: 369 tgctgctggcgggcgccag 169 188 SEQ ID NO: 1372 ctggatgtaaccaccagca 11178 11197 1 4 SEQ ID NO: 370 ctggtctgtccacaagatg 219 238 SEQ ID NO: 1373 catcctgaagaccagccag 380 399 1 4 SEQ ID NO: 371 ctgagagttccagtggagt 283 302 SEQ ID NO: 1374 actcaccctggacattcag 3383 3402 1 4 SEQ ID NO: 372 tccagtggagtccctggga 291 310 SEQ ID NO: 1375 tcccggagccaaggctgga 2675 2894 1 4 SEQ ID NO: 373 aggttgagctggaggttcc 348 365 SEQ ID NO: 1376 ggaaccctctccctcacct 4728 4747 1 4 SEQ ID NO: 374 tgagctggaggttccccag 350 369 SEQ ID NO: 1377 ctgggaggcatgatgctca 9163 9182 1 4 SEQ ID NO: 375 tctgcagcttcatcctgaa 370 389 SEQ ID NO: 1378 ttcaaatataatcggcaga 3261 3280 1 4 SEQ ID NO: 376 gccagtgcaccctgaaaga 394 413 SEQ ID NO: 1379 tcaccgttcttataatggc 5794 5813 1 4 SEQ ID NO: 377 ctctgaggagtttgctgca 464 483 SEQ ID NO: 1380 tgcaagaatattttgagag 6340 6359 1 4 SEQ ID NO: 378 aggtatgagctcaagctgg 492 511 SEQ ID NO: 1381 ccagtttccggggaaacct 12716 12735 1 4 SEQ ID NO: 379 tcctttacccggagaaaga 535 554 SEQ ID NO: 1382 tctttttgggaagcaagga 2219 2238 1 4 SEQ ID NO: 380 catcaagaggggcatcatt 575 594 SEQ ID NO: 1383 aatggtcaagttcctgatg 2277 2296 1 4 SEQ ID NO: 381 tcctggttcccccagagac 601 620 SEQ ID NO: 1384 gtctctgaactcagaagga 13988 14007 1 4 SEQ ID NO: 382 cagaagccaagcaagtgtt 622 641 SEQ ID NO: 1385 aacaaataaatggagtctt 14072 14091 1 4 SEQ ID NO: 383 aagcaagtgttgtttctgg 630 649 SEQ ID NO: 1386 ccagagccaggtcgagctt 11042 11061 1 4 SEQ ID NO: 384 tctggataccgtgtatgga 644 663 SEQ ID NO: 1387 tccatgtcccatttacaga 11356 11375 1 4 SEQ ID NO: 385 ccactcactttaccgtcaa 670 689 SEQ ID NO: 1388 ttgattttaacaaaagtgg 6817 6836 1 4 SEQ ID NO: 386 aggaagggcaatgtggcaa 693 712 SEQ ID NO: 1389 ttgcaagcaagtcttactt 3005 3024 1 4 SEQ ID NO: 387 gcaatgtggcaacagaaat 700 719 SEQ ID NO: 1390 atttccataccccgtttgc 3480 3499 1 4 SEQ ID NO: 388 caatgtggcaacagaaata 701 720 SEQ ID NO: 1391 tattcttcttttccaattg 13826 13845 1 4 SEQ ID NO: 389 tggcaacagaaatatccac 706 725 SEQ ID NO: 1392 gtggcttcccatattgcca 1887 1906 1 4 SEQ ID NO: 390 agagacctgggccagtgtg 729 748 SEQ ID NO: 1393 cacattacatttggtctct 2930 2949 1 4 SEQ ID NO: 391 tgtgatcgcttcaagccca 744 763 SEQ ID NO: 1394 tgggaaagccgccctcaca 5210 5229 1 4 SEQ ID NO: 392 gtgatcgcttcaagcccat 745 764 SEQ ID NO: 1395 atgggaaagccgccctcac 5209 5228 1 4 SEQ ID NO: 393 cagcccacttgctctcatc 776 795 SEQ ID NO: 1396 gatgctgaacagtgagctg 8144 8163 1 4 SEQ ID NO: 394 gctctcatcaaaggcatga 788 805 SEQ ID NO: 1397 tcataacagtactgtgagc 10337 10358 1 4 SEQ ID NO: 395 ccttgtcaactctgatcag 811 830 SEQ ID NO: 1398 ctgagtgggtttatcaagg 12445 12464 1 4 SEQ ID NO: 396 cttgtcaactctgatcagc 812 831 SEQ ID NO: 1399 gctgagtgggtttatcaag 12444 12483 1 4 SEQ ID NO: 397 agccatctgcaaggagcaa 864 903 SEQ ID NO: 1400 ttgcaatgagctcatggct 3805 3824 1 4 SEQ ID NO: 398 gccatctucaaggagcaac 885 904 SEQ ID NO: 1401 gttgcaatgagctcatggc 3804 3823 1 4 SEQ ID NO: 399 cttcctgcctttctcctac 908 927 SEQ ID NO: 1402 gtaggaataaatggagaag 9453 9472 1 4 SEQ ID NO: 400 ctttctcctacaagaataa 918 935 SEQ ID NO: 1403 ttattgctgaatccaaaag 13648 13667 1 4 SEQ ID NO: 401 gatcaacagccgcttcttt 989 1008 SEQ ID NO: 1404 aaagccatcactgatgatc 1661 1680 1 4 SEQ ID NO: 402 atcaacagccgcttctttg 990 1009 SEQ ID NO: 1405 caaagccatcactgatgat 1660 1679 1 4 SEQ ID NO: 403 acagccgcttctttggtga 994 1013 SEQ ID NO: 1406 tcacaaatcctttggctgt 9667 9686 1 4 SEQ ID NO: 404 aagatgggcctcgcatttg 1023 1042 SEQ ID NO: 1407 caaaatagaagggaatctt 2089 2088 1 4 SEQ ID NO: 405 tgttttgaagactctccag 1082 1101 SEQ ID NO: 1408 ctggtaactactttaaaca 5487 5506 1 4 SEQ ID NO: 406 ttgaagactctccaggaac 1086 1105 SEQ ID NO: 1409 gttcaatgaatttattcaa 13184 13203 1 4 SEQ ID NO: 407 aactgaaaaaactaaccat 1102 1121 SEQ ID NO: 1410 atggcattttttgcaagtt 14006 14025 1 4 SEQ ID NO: 408 ctgaaaaaactaaccatct 1104 1123 SEQ ID NO: 1411 agattgatgggcagttcag 4564 4583 1 4 SEQ ID NO: 409 aaaactaaccatctctgag 1109 1128 SEQ ID NO: 1412 ctcaaagaatgactttttt 2570 2589 1 4 SEQ ID NO: 410 tgagcaaaatatccagaga 1124 1143 SEQ ID NO: 1413 tctccagataaaaaactca 12201 12220 1 4 SEQ ID NO: 411 caataagctagttactgag 1154 1173 SEQ ID NO: 1414 ctcagatcaaagttaattg 12265 12284 1 4 SEQ ID NO: 412 tactgagctgagaggcctc 1166 1185 SEQ ID NO: 1415 gagggtagtcataacagta 10329 10348 1 4 SEQ ID NO: 413 gcctcagtgatgaagcagt 1180 1199 SEQ ID NO: 1416 actgttgactcaggaaggc 12572 12591 1 4 SEQ ID NO: 414 agtcacatctctcttgcca 1196 1215 SEQ ID NO: 1417 tggccacatagcatggact 8858 8877 1 4 SEQ ID NO: 415 atctctcttgccacagctg 1202 1221 SEQ ID NO: 1418 cagctgacctcatcgagat 2161 2180 1 4 SEQ ID NO: 416 tctctcttgccacagctga 1203 1222 SEQ ID NO: 1419 tcagctgacctcatcgaga 2160 2179 1 4 SEQ ID NO: 417 tgccacagctgattgaggt 1210 1229 SEQ ID NO: 1420 acctgcaccaaagctggca 13955 13974 1 4 SEQ ID NO: 418 gccacagctgattgaggta 1211 1230 SEQ ID NO: 1421 caccaaaaaccccaatggc 11240 11259 1 4 SEQ ID NO: 419 tcactttacaagccttggt 1240 1259 SEQ ID NO: 1422 accagatgctgaacagtga 8140 8159 1 4 SEQ ID NO: 420 cccttctgatagatgtggt 1324 1343 SEQ ID NO: 1423 accacttacagctagaggg 10816 10835 1 4 SEQ ID NO: 421 gtcacctacctggtggccc 1341 1380 SEQ ID NO: 1424 gggcgacctaagttgtgac 3431 3450 1 4 SEQ ID NO: 422 ccttgtatgcgctgagcca 1432 1451 SEQ ID NO: 1425 tggctggtaacctaaaagg 5578 5597 1 4 SEQ ID NO: 423 gacaaaccctccagggacc 1472 1491 SEQ ID NO: 1426 ggtcctttatgattatgtc 12347 12368 1 4 SEQ ID NO: 424 tgctaattacctgatggaa 1508 1527 SEQ ID NO: 1427 ttcccaaaagccatcagca 9930 9949 1 4 SEQ ID NO: 425 tgactgcactggggatgaa 1538 1557 SEQ ID NO: 1428 ttcaggtccatgcaagtca 10909 10928 1 4 SEQ ID NO: 426 actgcactggggatgacga 1540 1559 SEQ ID NO: 1429 tcttgaacacaaagtcagt 5999 8018 1 4 SEQ ID NO: 427 atgcagattacacctattt 1552 1571 SEQ ID NO: 1430 aaatgaaagtaaagatcat 8110 8129 1 4 SEQ ID NO: 428 accatggagcagttaactc 1602 1821 SEQ ID NO: 1431 gagtaaaccaaaacttggt 9018 9035 1 4 SEQ ID NO: 429 gcagttaactccagaactc 1610 1629 SEQ ID NO: 1432 gagttactgaaaaagctgc 13719 13738 1 4 SEQ ID NO: 430 cagaactcaagtcttcaat 1621 1640 SEQ ID NO: 1433 attggatatccaagatctg 1925 1944 1 4 SEQ ID NO: 431 caggctctgcggaaaatgg 1695 1714 SEQ ID NO: 1434 ccatgacctccagctcctg 2477 2496 1 4 SEQ ID NO: 432 ccaggaggttcttcttcag 1730 1749 SEQ ID NO: 1435 ctgaaatacactactctgg 5511 5530 1 4 SEQ ID NO: 433 ggttcttcttcagactttc 1736 1755 SEQ ID NO: 1436 gaaaaacttggaaacaacc 4431 4450 1 4 SEQ ID NO: 434 taccttgatgatgcttcta 1751 1770 SEQ ID NO: 1437 agaatccagatacaagaaa 8885 6904 1 4 SEQ ID NO: 435 ggagataagcgactgactg 1773 1792 SEQ ID NO: 1438 cagcatgcctagtttctcc 9944 9983 1 4 SEQ ID NO: 436 gctgcctatcttatgttga 1788 1807 SEQ ID NO: 1439 tcaatatcaaaagcccagc 12037 12056 1 4 SEQ ID NO: 437 actttgtggcttcccatat 1882 1901 SEQ ID NO: 1440 atatctggaaccttgaagt 10729 10748 1 4 SEQ ID NO: 438 gccaatatcttgaactcag 1902 1921 SEQ ID NO: 1441 ctgaactcagaaggatggc 13992 14011 1 4 SEQ ID NO: 439 aatatcttgaactcagaag 1905 1924 SEQ ID NO: 1442 cttccattctgaatatatt 13370 13389 1 4 SEQ ID NO: 440 ctcagaagaattggatatc 1916 1935 SEQ ID NO: 1443 gataaaagattactttgag 7265 7284 1 4 SEQ ID NO: 441 aagaattggatatccaaga 1921 1940 SEQ ID NO: 1444 tcttcaatttattcttctt 13817 13838 1 4 SEQ ID NO: 442 agaattggatatccaagat 1922 1941 SEQ ID NO: 1445 atcttcaatttattcttct 13816 13835 1 4 SEQ ID NO: 443 tggatatccaagatctgaa 1927 1948 SEQ ID NO: 1446 ttcacataccagaattcca 8317 8336 1 4 SEQ ID NO: 444 atatccaagatctgaaaaa 1930 1949 SEQ ID NO: 1447 tttttaaccagtcagatat 10177 10196 1 4 SEQ ID NO: 445 tatccaagatctgaaaaag 1931 1950 SEQ ID NO: 1448 ctttttaaccagtcagata 10178 10195 1 4 SEQ ID NO: 446 caagatctgaaaaagttag 1935 1954 SEQ ID NO: 1449 ctaaattcccatggtcttg 4965 4984 1 4 SEQ ID NO: 447 aagatctgaaaaagttagt 1936 1955 SEQ ID NO: 1450 actaaattcccatggtctt 4964 4983 1 4 SEQ ID NO: 448 tgaaaaagttagtgaaaga 1942 1961 SEQ ID NO: 1451 tctttctcgggaatattca 10822 10841 1 4 SEQ ID NO: 449 tccaactgtcatggacttc 1982 2001 SEQ ID NO: 1452 gaagcacatatgaactgga 13937 13956 1 4 SEQ ID NO: 450 tcagaaaattctctcggaa 1999 2018 SEQ ID NO: 1453 ttcctttaacaattcctga 9493 9512 1 4 SEQ ID NO: 451 ttccatcacttgacccagc 2044 2063 SEQ ID NO: 1454 gctgacatagggaatggaa 8433 8452 1 4 SEQ ID NO: 452 cccagcttcagccaaaata 2057 2076 SEQ ID NO: 1455 tattctatccaagattggg 7812 7831 1 4 SEQ ID NO: 453 agcctcagccaaaatagaa 2080 2079 SEQ ID NO: 1456 ttctatccaagattgggct 7814 7833 1 4 SEQ ID NO: 454 atcttatatttgatccaaa 2083 2102 SEQ ID NO: 1457 tttgaaaaacaaagcagat 11813 11832 1 4 SEQ ID NO: 455 tcttatatttgatccaaat 2084 2103 SEQ ID NO: 1458 attttttgcaagttaaaga 14011 14030 1 4 SEQ ID NO: 456 cttcctaaagaaagcatgc 2109 2128 SEQ ID NO: 1459 gcatggcattatgatgaag 3608 3825 1 4 SEQ ID NO: 457 ctaaagaaagcatgctgaa 2113 2132 SEQ ID NO: 1460 ttcagggtgtggagtttag 5686 5705 1 4 SEQ ID NO: 458 taaagaaagcatgctgaaa 2114 2133 SEQ ID NO: 1461 tttcttaaacattccttta 9482 9501 1 4 SEQ ID NO: 459 gagattggcttggaaggaa 2175 2194 SEQ ID NO: 1462 ttccctccattaagttctc 11701 11720 1 4 SEQ ID NO: 460 ctttgagccaacattggaa 2198 2217 SEQ ID NO: 1463 ttccaatgaccaagaaaag 11060 11079 1 4 SEQ ID NO: 461 cagacagtgtcaacaaagc 2245 2264 SEQ ID NO: 1464 gcttactggacgaactctg 8134 6153 1 4 SEQ ID NO: 462 cagtgtcaacaaagctttg 2249 2268 SEQ ID NO: 1465 caaattcctggatacactg 9849 9868 1 4 SEQ ID NO: 463 agtgtcaacaaagctttgt 2250 2269 SEQ ID NO: 1466 acacgaatacgtctacact 4351 4370 1 4 SEQ ID NO: 464 ctgatggtgtctctaaggt 2290 2309 SEQ ID NO: 1467 acctcggaacaatcctcag 3325 3344 1 4 SEQ ID NO: 465 tgatggtgtctctaaggtc 2291 2310 SEQ ID NO: 1468 gacctgcgcaacgagatca 8823 8842 1 4 SEQ ID NO: 466 aaacatgagcaggatatgg 2343 2382 SEQ ID NO: 1469 ccatgatctacatttgttt 8788 6807 1 4 SEQ ID NO: 467 gaagctgattaaagatttg 2387 2406 SEQ ID NO: 1470 caaaaacattttcaacttc 5279 5298 1 4 SEQ ID NO: 468 aaagatttgaaatccaaag 2397 2416 SEQ ID NO: 1471 ctttaagttcagcatcttt 7606 7625 1 4 SEQ ID NO: 469 gatgggtgcccgcactctg 2510 2529 SEQ ID NO: 1472 cagatttgaggattccatc 7975 7994 1 4 SEQ ID NO: 470 gggatcccccagatgattg 2532 2651 SEQ ID NO: 1473 caatcacaagtcgattccc 9075 9094 1 4 SEQ ID NO: 471 ttttcttcactacatcttc 2585 2604 SEQ ID NO: 1474 gaagtgtcagtggcaaaaa 10374 10393 1 4 SEQ ID NO: 472 tcttcactacatcttcatg 2568 2607 SEQ ID NO: 1475 catggcattatgatgaaga 3807 3628 1 4 SEQ ID NO: 473 tacatcttcatggagaatg 2595 2814 SEQ ID NO: 1476 cattatggaggcccatgta 9437 9456 1 4 SEQ ID NO: 474 ttcatggagaatgcctttg 2601 2620 SEQ ID NO: 1477 caaaatcaactttaatgaa 8599 8618 1 4 SEQ ID NO: 475 tcatggagaatgcctttga 2602 2621 SEQ ID NO: 1478 tcaacacaatcttcaatga 13108 13127 1 4 SEQ ID NO: 476 tttgaactccccactggag 2616 2635 SEQ ID NO: 1479 ctccccaggacctttcaaa 9834 9853 1 4 SEQ ID NO: 477 ttgaactccccactggagc 2617 2636 SEQ ID NO: 1480 gctccccaggacctttcaa 9833 9852 1 4 SEQ ID NO: 478 tgaactccccactggagct 2618 2837 SEQ ID NO: 1481 agctccccaggacctttca 9832 9851 1 4 SEQ ID NO: 479 cacaggagctggattacag 2627 2646 SEQ ID NO: 1482 ctgtttctgagtcccagtg 9336 9355 1 4 SEQ ID NO: 480 actggagctggattacagt 2628 2647 SEQ ID NO: 1483 actgtttctgagtcccagt 9335 9354 1 4 SEQ ID NO: 481 agttgcaaatatcttcatc 2644 2663 SEQ ID NO: 1484 gatgatgccaaaatcaact 8591 6610 1 4 SEQ ID NO: 482 gttgcaaatatcttcatct 2645 2864 SEQ ID NO: 1485 agatgatgccaaaatcaac 6590 6609 1 4 SEQ ID NO: 483 aaatatcttcatctggagt 2650 2669 SEQ ID NO: 1486 actcagaaggatggcattt 13996 14015 1 4 SEQ ID NO: 484 taaaactggaagtagccaa 2695 2714 SEQ ID NO: 1487 ttggttacaggaggcttta 7892 7811 1 4 SEQ ID NO: 485 ggctgaactggtggcaaaa 2720 2739 SEQ ID NO: 1488 ttttcttttcagcccagcc 9220 9239 1 4 SEQ ID NO: 486 tgtggagtttgtgacaaat 2750 2769 SEQ ID NO: 1489 attttcaagcaaatgcaca 8530 8549 1 4 SEQ ID NO: 487 ttgtgacaaatatgggcat 2758 2777 SEQ ID NO: 1490 atgcgtctaccttacacaa 9513 9532 1 4 SEQ ID NO: 488 atgaacccccacttcttcc 2811 2830 SEQ ID NO: 1491 ggaagctgaagtttatcat 2869 2888 1 4 SEQ ID NO: 489 cttccacgagtcgggtctg 2825 2844 SEQ ID NO: 1492 cagagctatcactgggaag 5227 5246 1 4 SEQ ID NO: 490 gagtcgggtctggaggctc 2832 2851 SEQ ID NO: 1493 gagcttactggacgaactc 6132 8151 1 4 SEQ ID NO: 491 cctaaaagctgggaagctg 2858 2877 SEQ ID NO: 1494 cagactccccagccgtagg 12112 12131 1 4 SEQ ID NO: 492 agctgggaagctgaagttt 2864 2883 SEQ ID NO: 1495 aaactgttaatttacagct 5455 5474 1 4 SEQ ID NO: 493 ccagattagagctggaact 3100 3125 SEQ ID NO: 1496 agtttccggggaaacctgg 12718 12737 1 4 SEQ ID NO: 494 ggataccctgaagtttgta 3200 3219 SEQ ID NO: 1497 tacagtattctgaaaatcc 8386 8404 1 4 SEQ ID NO: 495 ctgaggctaccatgacatt 3244 3263 SEQ ID NO: 1498 aatgagctcatggcttcag 3809 3828 1 4 SEQ ID NO: 496 tgtccagtgaagtccaaat 3289 3308 SEQ ID NO: 1499 attttgagaggaatcgaca 6349 6368 1 4 SEQ ID NO: 497 aattccggattttgatgtt 3305 3324 SEQ ID NO: 1500 aacacatgaatcacaaatt 8930 8949 1 4 SEQ ID NO: 498 ttccggattttgatgttga 3307 3326 SEQ ID NO: 1501 tcaaaacgagcttcaggaa 13199 13218 1 4 SEQ ID NO: 499 cggaacaatcctcagagtt 3329 3348 SEQ ID NO: 1502 aacttgtacaactggtccg 4203 4222 1 4 SEQ ID NO: 500 tcctcagagttaatgatga 3337 3356 SEQ ID NO: 1503 tcatcaattggttacagga 7585 7804 1 4 SEQ ID NO: 501 ctcaccctggccattcaga 3384 3403 SEQ ID NO: 1504 tctgcagaacaatgctgag 12431 12450 1 4 SEQ ID NO: 502 cattcagaacaagaaaatt 3395 3414 SEQ ID NO: 1505 aattgactttgtagaaatg 8096 8115 1 4 SEQ ID NO: 503 actgaggtcgccctcatgg 3414 3433 SEQ ID NO: 1506 ccatgcaagtcagcccagt 10916 10935 1 4 SEQ ID NO: 504 ttatttccataccccgttt 3478 3497 SEQ ID NO: 1507 aaactgcctatattgataa 13872 13891 1 4 SEQ ID NO: 505 gtttgcaagcagaagccag 3493 3512 SEQ ID NO: 1508 ctggacttctcttcaaaac 5400 5419 1 4 SEQ ID NO: 506 tttgcaagcagaagccaga 3494 3513 SEQ ID NO: 1509 tctgggtgtcgacagcaaa 5264 5283 1 4 SEQ ID NO: 507 ttgcaagcagaagccagaa 3495 3514 SEQ ID NO: 1510 ttctgggtgtcgacagcaa 5263 5282 1 4 SEQ ID NO: 508 ctgcttctccaaatggact 3548 3565 SEQ ID NO: 1511 agtcaagattgatgggcag 4559 4578 1 4 SEQ ID NO: 509 tgctacagcttatggctcc 3589 3588 SEQ ID NO: 1512 ggaggctttaagttcagca 7601 7620 1 4 SEQ ID NO: 510 acagcttatggctccacag 3573 3592 SEQ ID NO: 1513 ctgtatagcaaattcctgt 5889 5908 1 4 SEQ ID NO: 511 tttccaagagggtggcatg 3592 3611 SEQ ID NO: 1514 catggacttcttctggaaa 8869 8888 1 4 SEQ ID NO: 512 ccaagagggtggcatggca 3595 3614 SEQ ID NO: 1515 tgcccagcaagcaagttgg 9353 9372 1 4 SEQ ID NO: 513 gtggcatggcattatgatg 3603 3622 SEQ ID NO: 1516 catccttaacaccttccac 8063 8082 1 4 SEQ ID NO: 514 tgatgaagagaagattgaa 3617 3636 SEQ ID NO: 1517 ttcactgttcctgaaatca 7863 7882 1 4 SEQ ID NO: 515 gaagagaagcttgaatttg 3821 3640 SEQ ID NO: 1518 caaaaacattttcaacttc 5279 5298 1 4 SEQ ID NO: 516 gagaagattgaatttgaat 3824 3643 SEQ ID NO: 1519 attcataatcccaactctc 8270 8289 1 4 SEQ ID NO: 517 tttgaatggaacacaggca 3638 3655 SEQ ID NO: 1520 tgcctttgtgtacaccaaa 11228 11247 1 4 SEQ ID NO: 518 aggcaccactgtagatacc 3650 3669 SEQ ID NO: 1521 ggtaacctaaaaggagcct 5583 5602 1 4 SEQ ID NO: 519 caaaaaaatgacttccaat 3868 3687 SEQ ID NO: 1522 attgaagtacctacttttg 8358 8377 1 4 SEQ ID NO: 520 aaaaaaatgacttccaatt 3669 3688 SEQ ID NO: 1523 aattgaagtacctactttt 8357 8376 1 4 SEQ ID NO: 521 aaaaaatgacttcccattt 3670 3689 SEQ ID NO: 1524 aaatccaatctcctctttt 8398 8417 1 4 SEQ ID NO: 522 cagagtccctcaaacagac 3752 3771 SEQ ID NO: 1525 gtctgtgggattccatctg 4082 4101 1 4 SEQ ID NO: 523 ccattaatagttgcaatga 3795 3814 SEQ ID NO: 1526 tcataagttcaatgaattt 13178 13197 1 4 SEQ ID NO: 524 ttcaacctccagaacatgg 3891 3910 SEQ ID NO: 1527 ccattgaccagatgctgaa 8134 8153 1 4 SEQ ID NO: 525 tgggattgccagacttcca 3907 3926 SEQ ID NO: 1528 tggaaatgggcctgcccca 8895 8914 1 4 SEQ ID NO: 526 cagtttgaaaattgagatt 3988 4005 SEQ ID NO: 1529 aatcaccactcctccactg 9533 9552 1 4 SEQ ID NO: 527 gaaaattgagattcctttg 3992 4011 SEQ ID NO: 1530 caaaactaccacacatttc 13688 13705 1 4 SEQ ID NO: 528 tttgccttttggtggcaaa 4007 4026 SEQ ID NO: 1531 tttgagaggaatcgacaaa 6351 6370 1 4 SEQ ID NO: 529 ctccagagatctaaagatg 4028 4047 SEQ ID NO: 1532 catcaattggttacaggag 7586 7605 1 4 SEQ ID NO: 530 tctaaagatgttagagact 4037 4056 SEQ ID NO: 1533 agtccttcatgtccctaga 10025 10044 1 4 SEQ ID NO: 531 ctgtgggattccatctgcc 4084 4103 SEQ ID NO: 1534 ggcattttgaaaaaaacag 9727 12737 1 4 SEQ ID NO: 532 atctgccatctcgagagtt 4096 4115 SEQ ID NO: 1535 aactctcaaaccctaagat 8548 8567 1 4 SEQ ID NO: 533 tctcgagagttccaagtcc 4104 4123 SEQ ID NO: 1536 ggacattcctctagcgaga 8207 8226 1 4 SEQ ID NO: 534 agtccctacttttaccatt 4118 4137 SEQ ID NO: 1537 aatgaatacagccaggact 6078 6097 1 4 SEQ ID NO: 535 acttttaccattcccaagt 4125 4144 SEQ ID NO: 1538 actttgtagaaatgaaagt 8101 8120 1 4 SEQ ID NO: 536 cattcccaagttgtatcaa 4133 4152 SEQ ID NO: 1539 ttgaaggacttcaggaatg 12001 12020 1 4 SEQ ID NO: 537 accacatgaaggctgactc 4276 4295 SEQ ID NO: 1540 gagtaaaccaaaacttggt 9016 9035 1 4 SEQ ID NO: 538 tttcctacaatgtgcaagg 4309 4328 SEQ ID NO: 1541 cctttaacaattcctgaaa 9495 9514 1 4 SEQ ID NO: 539 ctggagaaacaacatatga 4330 4349 SEQ ID NO: 1542 tcattctgggtctttccag 11027 12450 1 4 SEQ ID NO: 540 atcatgtgatgggtctcta 4370 4389 SEQ ID NO: 1543 tagaattacagaaaatgat 6557 6576 1 4 SEQ ID NO: 541 catgtgatgggtctctacg 4372 4391 SEQ ID NO: 1544 cgtaggcaccgtgggcatg 12125 10935 1 4 SEQ ID NO: 542 ttctagattcgaatatcaa 4399 4418 SEQ ID NO: 1545 ttgatgatgctgtcaagaa 7300 13891 1 4 SEQ ID NO: 543 tggggaccacagatgtctg 4491 4510 SEQ ID NO: 1546 cagaattccagcttcccca 8326 5419 1 4 SEQ ID NO: 544 ctaacactggccggctcaa 4636 4655 SEQ ID NO: 1547 ttgaggctattgatgttag 6976 5283 1 4 SEQ ID NO: 545 taacactggccggctcaat 4637 4656 SEQ ID NO: 1548 attgaggctattgatgtta 6975 5282 1 4 SEQ ID NO: 546 aacactggccggctcaatg 4638 4657 SEQ ID NO: 1549 cattgaggctattgatgtt 6974 4578 1 4 SEQ ID NO: 547 ctggccggctcaatggaga 4642 4661 SEQ ID NO: 1550 tctccatctgcgctaccag 12065 7620 1 4 SEQ ID NO: 548 agataacaggaagatatga 4705 4724 SEQ ID NO: 1551 tcatctcctttcttcatct 10202 5908 1 4 SEQ ID NO: 549 tccctcacctccacctctg 4737 4756 SEQ ID NO: 1552 cagatatatatctcaggga 8176 8888 1 4 SEQ ID NO: 550 agctgactttaaaatctga 4810 4829 SEQ ID NO: 1553 tcaggctcttcagaaagct 7922 9372 1 4 SEQ ID NO: 551 ctgactttaaaatctgaca 4812 4831 SEQ ID NO: 1554 tgtcaagataaacaatcag 8732 8082 1 4 SEQ ID NO: 552 caagatggatatgaccttc 4865 4884 SEQ ID NO: 1555 gaagtagtactgcatcttg 6835 7882 1 4 SEQ ID NO: 553 gctgcgttctgaatatcag 4901 4920 SEQ ID NO: 1556 ctgagtcccagtgcccagc 9342 5298 1 4 SEQ ID NO: 554 cgttctgaatatcaggctg 4905 4924 SEQ ID NO: 1557 cagcaagtacctgagaacg 8603 8289 1 4 SEQ ID NO: 555 aattcccatggtcttgagt 4968 4987 SEQ ID NO: 1558 actcagatcaaagttaatt 12264 11247 1 4 SEQ ID NO: 556 tggtcttgagttaaatgct 4976 4995 SEQ ID NO: 1559 agcacagtacgaaaaacca 10801 5602 1 4 SEQ ID NO: 557 cttgagttaaatgctgaca 4980 4999 SEQ ID NO: 1560 tgtccctagaaatctcaag 10034 8377 1 4 SEQ ID NO: 558 ttgagttaaatgctgacat 4981 5000 SEQ ID NO: 1561 atgtccctagaaatctcaa 10033 8376 1 4 SEQ ID NO: 559 tgagttaaatgctgacatc 4982 5001 SEQ ID NO: 1562 gatggaaccctctccctca 4725 8417 1 4 SEQ ID NO: 560 acttgaagtgtagtctcct 5066 5105 SEQ ID NO: 1563 aggaaactcagatcaaagt 12259 4101 1 4 SEQ ID NO: 561 agtgtagtctcctggtgct 5092 5111 SEQ ID NO: 1564 agcagccagtggcaccact 12506 13197 1 4 SEQ ID NO: 562 gtgctggagaatgagctga 5106 5125 SEQ ID NO: 1565 tcagccaggtttatagcac 7726 8153 1 4 SEQ ID NO: 563 ctggggcatctatgaaatt 5143 5162 SEQ ID NO: 1566 aatttctgattaccaccag 13571 8914 1 4 SEQ ID NO: 564 atggccgcttcagggaaca 5170 5189 SEQ ID NO: 1567 tgttttttggaaatgccat 8641 9552 1 4 SEQ ID NO: 565 ttcagtctggatgggaaag 5199 5218 SEQ ID NO: 1568 ctttgacaggcattttgaa 9719 13705 1 4 SEQ ID NO: 566 ccatgattctaggtgtcga 5257 5276 SEQ ID NO: 1569 tcgatgcacatacaaatgg 5830 5849 1 4 SEQ ID NO: 567 aaaacattttcaacttcaa 5281 5300 SEQ ID NO: 1570 ttgatgttagagtgctttt 6985 7004 1 4 SEQ ID NO: 568 cttaagctctcaaatgaca 5318 5335 SEQ ID NO: 1571 tgtcctacaacaagttaag 7247 7266 1 4 SEQ ID NO: 569 ttaagctctcaaatgacat 5317 5336 SEQ ID NO: 1572 atgtcctacaacaagttaa 7248 7285 1 4 SEQ ID NO: 570 catgatgggctcatatgct 5333 5352 SEQ ID NO: 1573 agcatctttggctcacatg 7618 7635 1 4 SEQ ID NO: 571 tgggctcatatgctgaaat 5338 5357 SEQ ID NO: 1574 atttatcaaaagaagccca 12934 12953 1 4 SEQ ID NO: 572 actggacttctcttcaaaa 5399 5418 SEQ ID NO: 1575 ttttggcaagctatacagt 8372 8381 1 4 SEQ ID NO: 573 acttctcttcaaaacttga 5404 5423 SEQ ID NO: 1576 tcaattgggagagacaagt 6496 8515 1 4 SEQ ID NO: 574 ctgacaagttttataagca 5437 5458 SEQ ID NO: 1577 tgctttgtgagtttatcag 9885 9704 1 4 SEQ ID NO: 575 aagttttataagcaaactg 5442 5481 SEQ ID NO: 1578 cagtcatgtagaaaaactt 4421 4440 1 4 SEQ ID NO: 576 ctgttaatttacagctaca 5458 5477 SEQ ID NO: 1579 tgtactggaaaacgtacag 8380 8399 1 4 SEQ ID NO: 577 ttacagctacagccctatt 5466 5485 SEQ ID NO: 1580 aatattgatcaatttgtaa 6417 6436 1 4 SEQ ID NO: 578 tctggtaactactttaaac 5486 5505 SEQ ID NO: 1581 gtttgaaaaacaaagcaga 11812 11831 1 4 SEQ ID NO: 579 tttaaacagtgacctgaaa 5498 5517 SEQ ID NO: 1582 tttcatttgaaagaataaa 7024 7043 1 4 SEQ ID NO: 580 ttaaacagtgacctgaaat 5499 5518 SEQ ID NO: 1583 atttcaagcaagaacttaa 10426 10445 1 4 SEQ ID NO: 581 cagtgacctgaaatacaat 5504 5523 SEQ ID NO: 1584 attgacgtggagcttactg 6123 8142 1 4 SEQ ID NO: 582 tgtggctgataacctaaaa 5578 5595 SEQ ID NO: 1585 ttttgctggagaagccaca 10757 10778 1 4 SEQ ID NO: 583 ttatcagcaagctataaag 5649 5668 SEQ ID NO: 1586 ctttgcactatattcataa 12758 12775 1 4 SEQ ID NO: 584 ggttcagggtgtggagttt 5684 5703 SEQ ID NO: 1587 aaacacctaagagtaaacc 9006 9025 1 4 SEQ ID NO: 585 attcagactcactgcattt 5767 5788 SEQ ID NO: 1588 aaatgctgacatagggaat 8429 8448 1 4 SEQ ID NO: 586 ttcagactcactgcatttc 5768 5787 SEQ ID NO: 1589 gaaatattatgaacttgaa 13304 13323 1 4 SEQ ID NO: 587 tacaaatggcaatgggaaa 5840 5859 SEQ ID NO: 1590 tttcctaaagctggatgta 11168 11187 1 4 SEQ ID NO: 588 gctgtatagcaaattcctg 5888 5907 SEQ ID NO: 1591 caggtccatgcaagtcagc 10911 10930 1 4 SEQ ID NO: 589 tgagcagacaggcacctgg 6035 6054 SEQ ID NO: 1592 ccagcattcccacatctca 8333 8352 1 4 SEQ ID NO: 590 ggcacctggaaactcaaga 6045 6084 SEQ ID NO: 1593 tcttcgtgtttcaactgcc 11213 11232 1 4 SEQ ID NO: 591 tgaatacagccaggacttg 6080 6099 SEQ ID NO: 1594 caagtaagtgctaggttca 9372 9391 1 4 SEQ ID NO: 592 gaatacagccaggacttgg 6081 6100 SEQ ID NO: 1595 ccaacacttacttgaattc 10660 10879 1 4 SEQ ID NO: 593 ctggacgaactctggctga 6139 6158 SEQ ID NO: 1596 tcagaaagctaccttccag 7931 7950 1 4 SEQ ID NO: 594 ttttactcagtgagcccat 6193 6212 SEQ ID NO: 1597 atggacttcttctggaaaa 8870 8889 1 4 SEQ ID NO: 595 gatgagagatgccgttgag 6233 6252 SEQ ID NO: 1598 ctcatctcctttcttcatc 10201 10220 1 4 SEQ ID NO: 596 aattgttgcttttgtaaag 6269 6288 SEQ ID NO: 1599 cttttctaaacttgaaatt 9056 9075 1 4 SEQ ID NO: 597 cttttgtaaagtatgataa 6277 6296 SEQ ID NO: 1600 ttatgaacttgaagaaaag 13310 13329 1 4 SEQ ID NO: 598 tttgtaaagtatgataaaa 6279 6298 SEQ ID NO: 1601 ttttcacattagatgcaaa 8413 8432 1 4 SEQ ID NO: 599 tccattaacctcccatttt 6312 6331 SEQ ID NO: 1602 aaaattgatgatatctgga 10719 10738 1 4 SEQ ID NO: 600 ccagctctgcagcttcatc 6313 6332 SEQ ID NO: 1603 aaaagggtcatggaaatgg 8885 8904 1 4 SEQ ID NO: 601 agcttcatcctgaagacca 6338 6357 SEQ ID NO: 1604 ctcaattttgattttcaag 8520 8539 1 4 SEQ ID NO: 602 agaatattttgagaggaat 6344 6383 SEQ ID NO: 1605 attccctccattaagttct 11700 11719 1 4 SEQ ID NO: 603 attatagttgtactggaaa 6372 6391 SEQ ID NO: 1606 tttcaagcaagaacttaat 10427 10448 1 4 SEQ ID NO: 604 gaagcacatcaatattgat 6407 6426 SEQ ID NO: 1607 atcagttcagataaacttc 7991 8010 1 4 SEQ ID NO: 605 acatcaatattgatcaatt 6412 6431 SEQ ID NO: 1608 aattccctgaagttgatgt 11479 11498 1 4 SEQ ID NO: 606 gaaaactcccacagcaagc 6457 6476 SEQ ID NO: 1609 gctttctcttccacatttc 10052 10071 1 4 SEQ ID NO: 607 ctgaattcattcaattggg 6486 6505 SEQ ID NO: 1610 cccatttacagatcttcag 11363 11382 1 4 SEQ ID NO: 608 tgaattcattcaattggga 6487 6506 SEQ ID NO: 1611 tcccatttacagatcttca 11302 11381 1 4 SEQ ID NO: 609 aactgactgctctcacaaa 6532 6551 SEQ ID NO: 1612 tttgaggattccatcagtt 7979 7998 1 4 SEQ ID NO: 610 aaacgtatagaattacaga 6550 6589 SEQ ID NO: 1613 tctggctccctcaactttt 9042 9081 1 4 SEQ ID NO: 611 atcaactttaatgaaaaac 6603 6622 SEQ ID NO: 1614 gtttattgaaaatattgat 6803 6822 1 4 SEQ ID NO: 612 tgatttgaaaatagctatt 6686 6705 SEQ ID NO: 1615 aatattattgatgaaatca 6708 8727 1 4 SEQ ID NO: 613 atttgaaaatagctattgc 6688 6707 SEQ ID NO: 1616 gcaagaacttaatggaaat 10433 10452 1 4 SEQ ID NO: 614 attgctaatattattgatg 6702 6721 SEQ ID NO: 1617 catcacactgaataccaat 10151 10170 1 4 SEQ ID NO: 615 gaaaaattaaaaagtcttg 6729 6748 SEQ ID NO: 1618 caagagcttatgggatttc 11153 11172 1 4 SEQ ID NO: 616 actatcatatccgtgtaat 6754 6773 SEQ ID NO: 1619 attactttgagaaattagt 7273 7292 1 4 SEQ ID NO: 617 tattgattttaacaaaagt 6815 6834 SEQ ID NO: 1620 acttgacttcagagaaata 11396 11415 1 4 SEQ ID NO: 618 ctgcagcagcttaagagac 6906 6925 SEQ ID NO: 1621 gtcttcagtgaagatgcag 10691 10710 1 4 SEQ ID NO: 619 aaaacaacacattgaggct 6965 6984 SEQ ID NO: 1622 agcctcacctattactttt 10563 10582 1 4 SEQ ID NO: 620 ttgagcatgtcaaacactt 7051 7070 SEQ ID NO: 1623 aagtagctgagaaaatcaa 7096 7115 1 4 SEQ ID NO: 621 tttgaagtagctgagaaaa 7092 7111 SEQ ID NO: 1624 ttttcacattagatgcaaa 8413 8432 1 4 SEQ ID NO: 622 ttagtagagttggcccacc 7191 7210 SEQ ID NO: 1625 ggtggactcttgctgctaa 7768 7787 1 4 SEQ ID NO: 623 tgaaggagactattcagaa 7219 7238 SEQ ID NO: 1626 ttctcattttagattttca 8518 8537 1 4 SEQ ID NO: 624 gagactattcagaagctaa 7224 7243 SEQ ID NO: 1627 ttagccacagctctgtctc 10293 10312 1 4 SEQ ID NO: 625 acttagttggatttattga 7285 7304 SEQ ID NO: 1628 tcaagaagcttaatgaatt 7312 7331 1 4 SEQ ID NO: 626 gcttaatgaattatctttt 7319 7338 SEQ ID NO: 1629 aaaacgagcttcaggaagc 13201 13220 1 4 SEQ ID NO: 627 ttaacaaattccttgacat 7357 7376 SEQ ID NO: 1630 atgtcctacaacaagttaa 7246 7265 1 4 SEQ ID NO: 628 aaattaaagtcatttgatt 7386 7405 SEQ ID NO: 1631 aatcctttgacaggcattt 9715 9734 1 4 SEQ ID NO: 629 gactcaatggtgaaattca 7466 7475 SEQ ID NO: 1632 tgaaattcaatcacaagtc 9068 9087 1 4 SEQ ID NO: 630 gaaattcaggctctggaac 7467 7486 SEQ ID NO: 1633 gttctcaattttgattttc 8517 8536 1 4 SEQ ID NO: 631 actaccacaaaaagctgaa 7484 7503 SEQ ID NO: 1634 ttcaggaactattgctagt 10837 10656 1 4 SEQ ID NO: 632 ccaaaataaccttaatcat 7570 7589 SEQ ID NO: 1635 atgatttccctgaccttgg 10942 10981 1 4 SEQ ID NO: 633 aaataaccttaatcatcaa 7573 7592 SEQ ID NO: 1636 ttgaagtaaaagaaaattt 10741 10760 1 4 SEQ ID NO: 634 tttaagttcagcatctttg 7607 7626 SEQ ID NO: 1637 caaatctggatttcttaaa 9472 9491 1 4 SEQ ID NO: 635 caggtttatagcacacttg 7731 7750 SEQ ID NO: 1638 caagggttcactgttcctg 7857 7876 1 4 SEQ ID NO: 636 gttcactgttcctgaaatc 7862 7881 SEQ ID NO: 1639 gattctcagatgagggaac 8914 8933 1 4 SEQ ID NO: 637 cactgttcctgaaatcaag 7885 7884 SEQ ID NO: 1640 cttgaacacaaagtcagtg 6000 6019 1 4 SEQ ID NO: 638 actgttcctgaaatcaaga 7866 7885 SEQ ID NO: 1641 tcttgaacacaaagtcagt 5999 6018 1 4 SEQ ID NO: 639 gcctgcctttgaagtcagt 7901 7920 SEQ ID NO: 1642 actgttgactcaggaaggc 12572 12591 1 4 SEQ ID NO: 640 taacagatttgaggattcc 7972 7991 SEQ ID NO: 1643 ggaagcttctcaagagtta 13214 13233 1 4 SEQ ID NO: 641 gttttccacaccagaattt 8042 8061 SEQ ID NO: 1644 aaatttctctgctggaaac 9410 9429 1 4 SEQ ID NO: 642 tcagaaccattgaccagat 8128 8147 SEQ ID NO: 1645 atctgcagaacaatgctga 12430 12449 1 4 SEQ ID NO: 643 tagcgagaatcaccctgcc 8218 8237 SEQ ID NO: 1646 ggcagcttctggcttgcta 12293 12312 1 4 SEQ ID NO: 644 ccttaatgattttcaagtt 8281 8310 SEQ ID NO: 1647 aactgttgactcaggcagg 12571 12590 1 4 SEQ ID NO: 645 acataccagaattccagct 8320 8339 SEQ ID NO: 1648 agctgccagtccttcatgt 10018 10037 1 4 SEQ ID NO: 646 aatgctgacatagggaatg 8430 8449 SEQ ID NO: 1649 cattaatactgccatcatt 9997 10016 1 4 SEQ ID NO: 647 atgctgacatagggaatgg 8431 8450 SEQ ID NO: 1650 ccatttgagatcacggcat 9237 9256 1 4 SEQ ID NO: 648 aaccacctcagcaaacgaa 8450 8489 SEQ ID NO: 1651 ttcgttttccattaaggtt 9283 9302 1 4 SEQ ID NO: 649 agcaggtatcgcagcttcc 8468 8487 SEQ ID NO: 1652 ggaagtggccctgaatgct 10984 10983 1 4 SEQ ID NO: 650 tgcacaactctcaaaccct 8543 8562 SEQ ID NO: 1653 agggaaagagaagattgca 13493 13512 1 4 SEQ ID NO: 651 aggagtcagtgaagttctc 8584 8603 SEQ ID NO: 1654 gagaacttactatcatcct 13780 13799 1 4 SEQ ID NO: 652 tttttggaaatgccattga 8644 8663 SEQ ID NO: 1655 tcaatgaatttattcaaaa 13186 13205 1 4 SEQ ID NO: 653 aatggagtgattgtcaaga 8721 8740 SEQ ID NO: 1656 tcttttcagcccagccatt 9223 9242 1 4 SEQ ID NO: 654 gtcaagataaacaatcagc 8733 8762 SEQ ID NO: 1657 gctgactttaaaatctgac 4811 4830 1 4 SEQ ID NO: 655 tccacaaattgaacatccc 8779 8798 SEQ ID NO: 1658 gggatttcctaaagctggc 11164 11183 1 4 SEQ ID NO: 656 ttgaacatccccaaactgg 8787 8806 SEQ ID NO: 1659 ccagtttccagggactcaa 12595 12614 1 4 SEQ ID NO: 657 acatccccaaactggactt 8791 8810 SEQ ID NO: 1660 aagtcgattcccagcatgt 9082 9101 1 4 SEQ ID NO: 658 acttctctagtcaggctga 8806 8825 SEQ ID NO: 1661 tcagatggaaaaatgaagt 11002 11021 1 4 SEQ ID NO: 659 tgaatcacaaattagtttc 8936 8955 SEQ ID NO: 1662 gaaagtccataatggttca 12809 12828 1 4 SEQ ID NO: 660 agaaggacccctcacttcc 8960 8979 SEQ ID NO: 1663 ggaagaagaggcagcttct 12284 12303 1 4 SEQ ID NO: 661 ttggactgtccaataagat 8980 8999 SEQ ID NO: 1664 atctaaatgcagtagccaa 11626 11645 1 4 SEQ ID NO: 662 actgtccaataagatcaat 8984 9003 SEQ ID NO: 1665 attgataaaaccatacagt 13883 13902 1 4 SEQ ID NO: 663 ctgtccaataagatcaata 8985 9004 SEQ ID NO: 1666 tattgataaaaccatacag 13882 13901 1 4 SEQ ID NO: 664 gtttatgaatctggctccc 9033 9052 SEQ ID NO: 1667 gggaatctgatgaggaaac 12247 12266 1 4 SEQ ID NO: 665 atgaatctggctccctcaa 9037 9056 SEQ ID NO: 1668 ttgagttgcccaccatcat 11659 11678 1 4 SEQ ID NO: 666 ctcaacttttctaaacttg 9051 9070 SEQ ID NO: 1669 caagatcgcagactttgag 11845 11864 1 4 SEQ ID NO: 667 ctaaaggcatggcactgtt 9121 9140 SEQ ID NO: 1670 aacagaaacaatgcattag 9741 9760 1 4 SEQ ID NO: 668 aaggcatggcactgtttgg 9124 9143 SEQ ID NO: 1671 ccaagaaaaggcacacctt 11069 11088 1 4 SEQ ID NO: 669 atccacaaacaatgaaggg 9254 9273 SEQ ID NO: 1672 ccctaacagatttgaggat 7969 7988 1 4 SEQ ID NO: 670 ggcatttgaaagttcgttt 9271 9290 SEQ ID NO: 1673 aaacaaacacaggcattcc 9647 9666 1 4 SEQ ID NO: 671 aataactatgcactgtttc 9324 9343 SEQ ID NO: 1674 gaaatactgttttcctatt 12828 12847 1 4 SEQ ID NO: 672 gaaacaacgagaacattat 9424 9443 SEQ ID NO: 1675 ataaactgcaagatttttc 13600 13619 1 4 SEQ ID NO: 673 ttcttgaaaacgacaaagc 9591 9610 SEQ ID NO: 1676 gctttccaatgaccaagaa 11057 11076 1 4 SEQ ID NO: 674 ataagaaaaacaaacacag 9640 9659 SEQ ID NO: 1677 ctgtgctttgtgagtttat 9682 9701 1 4 SEQ ID NO: 675 aaaacaaacacaggcattc 9646 9865 SEQ ID NO: 1678 gaatttgaaagttcgtttt 9272 9291 1 4 SEQ ID NO: 676 gcattccatcacaaatcct 9859 9678 SEQ ID NO: 1679 aggaagtggccctgaatgc 10963 10982 1 4 SEQ ID NO: 677 tttgaaaaaaacagaaaca 9732 9751 SEQ ID NO: 1680 tgttgaaagatttatcaaa 12925 12944 1 4 SEQ ID NO: 678 caatgcattagattttgtc 9749 9768 SEQ ID NO: 1681 gacaagaaaaaggggattg 10271 10290 1 4 SEQ ID NO: 679 caacgctgaaaaatctcag 9809 9828 SEQ ID NO: 1682 ctgagaacttcatcatttg 11430 11449 1 4 SEQ ID NO: 680 cctggatacactgttccag 9855 9874 SEQ ID NO: 1683 ctggacttctctagtcagg 8802 8821 1 4 SEQ ID NO: 681 gttgaagtgtctccattca 9882 9901 SEQ ID NO: 1684 tgaatctggctccctcaac 9038 9057 1 4 SEQ ID NO: 682 tttctccatcctaggttct 9956 9975 SEQ ID NO: 1685 agaatccagatacaagaaa 6885 6904 1 4 SEQ ID NO: 683 ttctccatcctaggttctg 9957 9976 SEQ ID NO: 1686 cagaatccagatacaagaa 6884 6903 1 4 SEQ ID NO: 684 tcattagagctgccagtcc 10011 10030 SEQ ID NO: 1687 ggacagtgaaatattatga 13297 13316 1 4 SEQ ID NO: 685 tgctgaactttttaaccag 10189 10188 SEQ ID NO: 1688 ctggatgtaaccaccagca 11178 11197 1 4 SEQ ID NO: 686 ctcctttcttcatcttcat 10206 10225 SEQ ID NO: 1689 atgaagcttgatccaggag 13784 13783 1 4 SEQ ID NO: 687 tgtcattgatgcactgcag 10226 10245 SEQ ID NO: 1690 ctgcgctaccagaaagaca 12072 12091 1 4 SEQ ID NO: 688 tgatgcactgcagtacaaa 10232 10251 SEQ ID NO: 1691 tttgagttgcccaccatca 11658 11677 1 4 SEQ ID NO: 689 agctctgtctctgagcaac 10301 10320 SEQ ID NO: 1692 gttgaccacaagcttagct 10539 10558 1 4 SEQ ID NO: 690 agccgaaattccaattttg 10400 10419 SEQ ID NO: 1693 caaagctggcaccagggct 13963 13982 1 4 SEQ ID NO: 691 ttgagaatgaatttcaagc 10416 10435 SEQ ID NO: 1694 gcttcaggcagcttctcaa 13208 13227 1 4 SEQ ID NO: 692 aaacctactgtctcttcct 10461 10480 SEQ ID NO: 1695 aggaaggccaagccagttt 12583 12802 1 4 SEQ ID NO: 693 tacttttccattgagtcat 10575 10594 SEQ ID NO: 1696 atgattatgtcaacaagta 12355 12374 1 4 SEQ ID NO: 694 tcaggtccatgcaagtcag 10910 10929 SEQ ID NO: 1697 ctgacatcttaggcactga 4993 5012 1 4 SEQ ID NO: 695 atgcaagtcagcccagttc 10918 10937 SEQ ID NO: 1698 gaactcagaaggatggcat 13994 14013 1 4 SEQ ID NO: 696 tgaatgctaacactaagaa 10975 10994 SEQ ID NO: 1699 ttctcaattttgattttca 8518 8537 1 4 SEQ ID NO: 697 agaagatcagatggaaaaa 10996 11015 SEQ ID NO: 1700 ttttctaaatggaacttct 12165 12184 1 4 SEQ ID NO: 698 ggctattcattctccatcc 11256 11275 SEQ ID NO: 1701 ggatctaaatgcagtagcc 11824 11643 1 4 SEQ ID NO: 699 aaagttttggctgataaat 11280 11299 SEQ ID NO: 1702 atttcttaaacattccttt 9481 9500 1 4 SEQ ID NO: 700 agttttggctgataaattc 11282 11301 SEQ ID NO: 1703 gaatctggctccctcaact 9039 9058 1 4 SEQ ID NO: 701 ctgggctgaaactaaatga 11308 11327 SEQ ID NO: 1704 tcattctgggtctttccag 11027 11046 1 4 SEQ ID NO: 702 cagagaaatacaaatctat 11405 11424 SEQ ID NO: 1705 atagcatggacttcttctg 8865 8884 1 4 SEQ ID NO: 703 gaggtaaaattccctgaag 11472 11491 SEQ ID NO: 1706 cttctggcttgctaacctc 12298 12317 1 4 SEQ ID NO: 704 cttttttgagataaccgtg 11537 11556 SEQ ID NO: 1707 cacggagttactgaaaaag 13715 13734 1 4 SEQ ID NO: 705 gctggaattgtcattcctt 11727 11746 SEQ ID NO: 1708 aaggcatctccacctcagc 12094 12113 1 4 SEQ ID NO: 706 gtgtataatgccacttgga 11787 11806 SEQ ID NO: 1709 tccaagatgagatcaacac 13096 13115 1 4 SEQ ID NO: 707 attccacatgcagctcaac 11851 11870 SEQ ID NO: 1710 gttgagaagccccaagaat 5246 6265 1 4 SEQ ID NO: 708 tgaagaagatggcaaattt 11984 12003 SEQ ID NO: 1711 aaattctcttttcttttca 9212 9231 1 4 SEQ ID NO: 709 atcaaaagcccagcgttca 12042 12081 SEQ ID NO: 1712 tgaaagtcacacatctgat 12881 12680 1 4 SEQ 1D NO: 710 gtgggcatggatatggatg 12135 12154 SEQ ID NO: 1713 catccttaacaccttccac 8063 8082 1 4 SEQ ID NO: 711 aaatggaacttctactaca 12171 12190 SEQ ID NO: 1714 tgtaccataagccatattt 10080 10099 1 4 SEQ ID NO: 712 aaaaactcaccatattcaa 12211 12230 SEQ ID NO: 1715 ttgatgttagagtgctttt 6985 7004 1 4 SEQ ID NO: 713 ctgagaagaaatctgcaga 12420 12439 SEQ ID NO: 1716 tatgcacagaaatattcag 13439 13458 1 4 SEQ ID NO: 714 acaatgctgagtgggttta 12439 12458 SEQ ID NO: 1717 taaatggagtctttattgt 14078 14097 1 4 SEQ ID NO: 715 caatgctgagtgggtttat 12440 12459 SEQ ID NO: 1718 ataaatggagtctttattg 14077 14096 1 4 SEQ ID NO: 716 ttaggcaaattgatgatat 12489 12488 SEQ ID NO: 1719 atattgtcagtgcctctaa 13384 13403 1 4 SEQ ID NO: 717 ataaactaatagatgtaat 12889 12908 SEQ ID NO: 1720 attactatgaaaaatttat 13633 13852 1 4 SEQ ID NO: 718 ccaactaatagaagataac 13031 13050 SEQ ID NO: 1721 gttattttgctaaacttgg 14044 14063 1 4 SEQ ID NO: 719 ttaattatatccaagatga 13087 13108 SEQ ID NO: 1722 tcatcctctaattttttaa 13792 13811 1 4 SEQ ID NO: 720 tttaaattgttgaaagaaa 13143 13162 SEQ ID NO: 1723 tttcatttgaaagaataaa 7024 7043 1 4 SEQ ID NO: 721 aagttcaatgaatttattc 13182 13201 SEQ ID NO: 1724 gaataccaatgctgaactt 10160 10179 1 4 SEQ ID NO: 722 ttgaagaaaagatagtcag 13318 13337 SEQ ID NO: 1725 ctgagagaagtgtcttcaa 12399 12418 1 4 SEQ ID NO: 723 acttccattctgaatatat 13369 13388 SEQ ID NO: 1726 atatctggaaccttgaagt 10729 10748 1 4 SEQ ID NO: 724 cacagaaatattcaggaat 13443 13462 SEQ ID NO: 1727 attccctgaagttgatgtg 11480 11499 1 4 SEQ ID NO: 725 ccattgcgacgaagaaaat 13552 13571 SEQ ID NO: 1728 atttttattcctgccatgg 10095 10114 1 4 SEQ ID NO: 726 tataaactgcaagattttt 13599 13618 SEQ ID NO: 1729 aaaattcaaactgcctata 13865 13884 1 4 SEQ ID NO: 727 tctgattactatgaaaaat 13629 13648 SEQ ID NO: 1730 atttgtaagaaaatacaga 6428 6447 1 4 SEQ ID NO: 728 ggagttactaaaaaagctg 13718 13737 SEQ ID NO: 1731 cagcatgcctagtttctcc 9944 9963 1 4 SEQ ID NO: 729 tgaagcttgctccaggaga 13765 13784 SEQ ID NO: 1732 tctcctttcttcatcttca 10205 10224 1 4 SEQ ID NO: 730 tgaactggacctgcaccaa 13947 13986 SEQ ID NO: 1733 ttggtagagcaagggttca 7848 7887 1 4 SEQ ID NO: 731 ttgctaaacttgggggagg 14050 4069 SEQ ID NO: 1734 cctcctacagtggtggcaa 4222 4241 1 4 SEQ ID NO: 732 gattcgaatatcaaattca 4404 4423 SEQ ID NO: 1735 tgaaaacgacaaagcaatc 9595 9614 3 3 SEQ ID NO: 733 atttgtttgtcaaagaagt 4543 4562 SEQ ID NO: 1736 acttttctaaacttgaaat 9055 9074 3 3 SEQ ID NO: 734 tctcggttgctgccgctga 25 44 SEQ ID NO: 1737 tcagcccagccatttgaga 9228 9247 2 3 SEQ ID NO: 735 gctgaggagcccgcccagc 39 58 SEQ ID NO: 1738 gctggatgtaaccaccagc 11177 11196 2 3 SEQ ID NO: 736 ctggtctgtccaaaagatg 219 238 SEQ ID NO: 1739 catcagaaccattgaccag 8126 8145 2 3 SEQ ID NO: 737 ctgagagttccagtggagt 283 302 SEQ ID NO: 1740 actcaatggtgaaattcag 7457 7476 2 3 SEQ ID NO: 738 cagtgcaccctgaaagagg 398 415 SEQ ID NO: 1741 cctcacttcctttggactg 8989 8988 2 3 SEQ ID NO: 739 ctctgaggagtttgctgca 464 483 SEQ ID NO: 1742 tgcaaacttgacttcagag 11391 11410 2 3 SEQ ID NO: 740 acatcaagaggggcatcat 574 593 SEQ ID NO: 1743 atgacgttcttgagcatgt 7042 7061 2 3 SEQ ID NO: 741 ctgatcagcagcagccagt 822 841 SEQ ID NO: 1744 actggacttctctagtcag 8801 8820 2 3 SEQ ID NO: 742 ggacgctaagaggaagcat 857 876 SEQ ID NO: 1745 atgcctacgttccatgtcc 11346 11365 2 3 SEQ ID NO: 743 agctgttttgaagactctc 1079 1098 SEQ ID NO: 1746 gagaagtgtcttcaaagct 12403 12422 2 3 SEQ ID NO: 744 tgaaaaacctaaccatctc 1105 1124 SEQ ID NO: 1747 gagatcaacacaatcttca 13104 13123 2 3 SEQ ID NO: 745 ctgagctgagaggcctcag 1168 1187 SEQ ID NO: 1748 ctgaattactgcacctcag 3027 3046 2 3 SEQ ID NO: 746 tgaaacgtgtgcatgccaa 1303 1322 SEQ ID NO: 1749 ttggtagagcaagggttca 7848 7867 2 3 SEQ ID NO: 747 ccttgtatgcgctgagcca 1432 1451 SEQ ID NO: 1750 tggcactgtttggagaagg 9130 9149 2 3 SEQ ID NO: 748 aggagctgctggacattgc 1492 1511 SEQ ID NO: 1751 gcaagtcagcccagttcct 10920 10939 2 3 SEQ ID NO: 749 atttgattctgcgggtcat 1567 1588 SEQ ID NO: 1752 atgaaaccaatgacaaaat 7420 7439 2 3 SEQ ID NO: 750 tccagaactcaagtcttca 1619 1638 SEQ ID NO: 1753 tgaaatacaatgctctgga 5512 5531 2 3 SEQ ID NO: 751 ggttcttcttcagactttc 1736 1755 SEQ ID NO: 1754 gaaataccaagtcaacacc 10447 10468 2 3 SEQ ID NO: 752 gttgatgaggagtccttca 1802 1821 SEQ ID NO: 1755 tgaaaaagctgcaatcaac 13728 13745 2 3 SEQ ID NO: 753 tcccagatctgaaaaagtt 1933 1952 SEQ ID NO: 1756 aactgcttctccaaatgga 3544 3563 2 3 SEQ ID NO: 754 agttagtgaaagaagttct 1948 1987 SEQ ID NO: 1757 agaattcataatcccaact 8267 8286 2 3 SEQ ID NO: 755 gaagggaatcttatatttg 2076 2095 SEQ ID NO: 1758 caaaacctactgtctcttc 10459 10478 2 3 SEQ ID NO: 756 ggaagctctttttgggaag 2213 2232 SEQ ID NO: 1759 cttcacataccagaattcc 8316 8335 2 3 SEQ ID NO: 757 tggaataatgctcagtgtt 2366 2385 SEQ ID NO: 1760 aacaaacacaggcattcca 9648 9887 2 3 SEQ ID NO: 758 gatttgaaatccaaagaag 2400 2419 SEQ ID NO: 1761 cttcatgtccctagaaatc 10029 10048 2 3 SEQ ID NO: 759 tccaaagaagtcccggaag 2409 2428 SEQ ID NO: 1762 cttcagcctgctttctgga 4943 4962 2 3 SEQ ID NO: 760 aggaagggctcaaagaatg 2582 2581 SEQ ID NO: 1763 cattagagctgccagtcct 10012 10031 2 3 SEQ ID NO: 761 agaatgacttttttcttca 2575 2594 SEQ ID NO: 1764 tgaagatgacgacttttct 12152 12171 2 3 SEQ ID NO: 762 tttgtgacaaatatgggca 2757 2778 SEQ ID NO: 1765 tgccagtttgaaaaacaaa 11807 11826 2 3 SEQ ID NO: 763 ctgaggctaccatgacatt 3244 3263 SEQ ID NO: 1766 aatgtcagctcttgttcag 10895 10914 2 3 SEQ ID NO: 764 gtagataccaaaaaaatga 3660 3679 SEQ ID NO: 1767 tcatttgccctcaacctac 11442 11481 2 3 SEQ ID NO: 765 aaatgacttccaatttccc 3673 3692 SEQ ID NO: 1768 gggaactgttgaaagattt 12919 12938 2 3 SEQ ID NO: 766 atgacttccaatttccctg 3675 3694 SEQ ID NO: 1769 caggagaacttactatcat 13777 13798 2 3 SEQ ID NO: 767 atctgccatctcgagagtt 4096 4115 SEQ ID NO: 1770 aactcctccactgaaagat 9539 9558 2 3 SEQ ID NO: 768 atttgtttgtcaaagaagt 4543 4562 SEQ ID NO: 1771 acttccgtttaccagaaat 8239 8258 2 3 SEQ ID NO: 769 gcagagcttggcctctctg 5127 5146 SEQ ID NO: 1772 cagagctttctgccactgc 13510 13529 2 3 SEQ ID NO: 770 atatgctgaaatgaaattt 5345 5364 SEQ ID NO: 1773 aaattcaaactgcctatat 13866 13885 2 3 SEQ ID NO: 771 tcaaaacttgacaacattt 5412 5431 SEQ ID NO: 1774 aaatacttccacaaattga 8772 8791 2 3 SEQ ID NO: 772 cagtgacctgaaatacaat 5504 5523 SEQ ID NO: 1775 attgaacatccccaaactg 8786 8805 2 3 SEQ ID NO: 773 tacaaatggcaatgggaaa 5840 5859 SEQ ID NO: 1776 ttttcaagcaaatgcacaa 11221 11240 2 3 SEQ ID NO: 774 cttttgtaaagtatgataa 6277 6296 SEQ ID NO: 1777 ttattgctgaatccaaaag 13648 13667 2 3 SEQ ID NO: 775 ttgtaaagtatgataaaaa 6260 6299 SEQ ID NO: 1778 ttttcaagcaaatgcacaa 8531 8550 2 3 SEQ ID NO: 776 tccattaacctcccatttt 6312 6331 SEQ ID NO: 1779 aaaagaaaattttgctgga 10748 10767 2 3 SEQ ID NO: 777 gattatctgaattcattca 6480 6499 SEQ ID NO: 1780 tgaagtagaccaacaaatc 7154 7173 2 3 SEQ ID NO: 778 aattgggagagacaagttt 6498 6517 SEQ ID NO: 1781 aaactaaatgatctaaatt 11316 11335 2 3 SEQ ID NO: 779 atttgaaaatagctattgc 6688 6707 SEQ ID NO: 1782 gcaatttctgcacagaaat 13433 13452 2 3 SEQ ID NO: 780 tgagcatgtcaaacacttt 7052 7071 SEQ ID NO: 1783 aaagccattcagtctctca 12963 12982 2 3 SEQ ID NO: 781 ttgaagatgttaacaaatt 7348 7367 SEQ ID NO: 1784 aattccatatgaaagtcaa 12652 12671 2 3 SEQ ID NO: 782 acttgtcacctacatttct 7745 7764 SEQ ID NO: 1785 agaatattttgatccaagt 13268 13287 2 3 SEQ ID NO: 783 gttttccacaccagaattt 8042 8081 SEQ ID NO: 1786 aaatctggatttcttaaac 9473 9492 2 3 SEQ ID NO: 784 ataagtacaaccaaaattt 9397 9418 SEQ ID NO: 1787 aaataaatggagtctttat 14075 14094 2 3 SEQ ID NO: 785 cgggacctgcggggctgag 0 19 SEQ ID NO: 1788 ctcagttaactgtgtcccg 11563 11582 1 3 SEQ ID NO: 786 agtgcccttctcggttgct 17 38 SEQ ID NO: 1789 agcatctgattgactcact 12670 12889 1 3 SEQ ID NO: 787 gctgaggagcccgcccagc 39 58 SEQ ID NO: 1790 gctgattgaggtgtccagc 1217 1236 1 3 SEQ ID NO: 788 gaggagcccgcccagccag 42 81 SEQ ID NO: 1791 ctggatcacagagtccctc 3744 3763 1 3 SEQ ID NO: 789 gggccgcgaggccgaggcc 84 83 SEQ ID NO: 1792 ggccctgatccccgagccc 1355 1374 1 3 SEQ ID NO: 790 ccaggccgcagcccaggag 81 100 SEQ ID NO: 1793 ctcccggagccaaggctgg 2874 2893 1 3 SEQ ID NO: 791 ggagccgccccaccgcagc 96 115 SEQ ID NO: 1794 gctgttttgaagactctcc 1080 1099 1 3 SEQ ID NO: 792 gaagaggaaatgctggaaa 192 211 SEQ ID NO: 1795 tttcaagttcctgaccttc 8301 8320 1 3 SEQ ID NO: 793 caaaagatgcgacccgatt 229 248 SEQ ID NO: 1796 aatcttattggggattttg 7077 7096 1 3 SEQ ID NO: 794 attcaagcacctccggaag 245 264 SEQ ID NO: 1797 cttccacatttcaaggaat 10059 10078 1 3 SEQ ID NO: 795 gttccagtggagtccctgg 289 308 SEQ ID NO: 1798 ccagcaagtacctgagaac 8602 8821 1 3 SEQ ID NO: 796 gactgctgattcaagaagt 308 327 SEQ ID NO: 1799 acttgaagaaaagatagtc 13316 13335 1 3 SEQ ID NO: 797 gtgccaccaggatcaactg 325 344 SEQ ID NO: 1800 cagtgaagctgcagggcac 10696 10715 1 3 SEQ ID NO: 798 gatcaactgcaaggttgag 335 354 SEQ ID NO: 1801 ctcacctccacctctgatc 4740 4759 1 3 SEQ ID NO: 799 actgcaaggttgagctgga 340 359 SEQ ID NO: 1802 tccactcacatcctccagt 1281 1300 1 3 SEQ ID NO: 800 ccagctctgcagcttcatc 365 384 SEQ ID NO: 1803 gatgtggtcacctacctgg 1335 1354 1 3 SEQ ID NO: 801 agcttcatcctgaagacca 375 394 SEQ ID NO: 1804 tggtgctggagaatgagct 5104 5123 1 3 SEQ ID NO: 802 cttcatcctgaagaccagc 377 396 SEQ ID NO: 1805 gctggagtaaaactggaag 2688 2707 1 3 SEQ ID NO: 803 ccagccagtgcaccctgaa 391 410 SEQ ID NO: 1806 ttcaagatgactgcactgg 1531 1550 1 3 SEQ ID NO: 804 cagtgcaccctgaaagagg 398 415 SEQ ID NO: 1807 cctcacagagctatcactg 5222 5241 1 3 SEQ ID NO: 805 tggcttcaaccctgagggc 419 438 SEQ ID NO: 1808 gcccactgatcgcctgcca 3525 3544 1 3 SEQ ID NO: 806 cttcaaccctgagggcaaa 422 441 SEQ ID NO: 1809 tttgagccaacattggaag 2199 2218 1 3 SEQ ID NO: 807 ttcaaccctgagggcaaag 423 442 SEQ ID NO: 1810 ctttgacaggcattttgaa 9719 9738 1 3 SEQ ID NO: 808 cttgctgaagaaaaccaag 443 482 SEQ ID NO: 1811 cttgaaattcaatcacaag 9068 9085 1 3 SEQ ID NO: 809 tgctgaagaaaaccaagaa 445 464 SEQ ID NO: 1812 ttctgctgccttatcagca 5639 5658 1 3 SEQ ID NO: 810 ttgctgcagccatgtccag 475 494 SEQ ID NO: 1813 ctggtcagtttgcaagcaa 2998 3015 1 3 SEQ ID NO: 811 tgctgcagccatgtccagg 476 495 SEQ ID NO: 1814 cctggtcagtttgcaagca 2995 3014 1 3 SEQ ID NO: 812 agccatgtccaggtatgag 482 501 SEQ ID NO: 1815 ctcacatcctccagtggct 1285 1304 1 3 SEQ ID NO: 813 agctcaagctggccattcc 499 518 SEQ ID NO: 1816 ggaactaccacaaaaagct 7481 7500 1 3 SEQ ID NO: 814 agaagggaagcaggttttc 518 537 SEQ ID NO: 1817 gaaatcttcaatttattct 13813 13832 1 3 SEQ ID NO: 815 aagggaagcaggttttcct 520 539 SEQ ID NO: 1818 aggacaccaaaataacctt 7564 7583 1 3 SEQ ID NO: 816 agaaagatgaacctactta 647 566 SEQ ID NO: 1819 taagaactttaccacttct 4844 4863 1 3 SEQ ID NO: 817 atcctgaacatcaagaggg 567 586 SEQ ID NO: 1820 ccctaacagatttgaggat 7969 7988 1 3 SEQ ID NO: 818 tcctgaacatcaagagggg 568 587 SEQ ID NO: 1821 cccctaacagatttgagga 7968 7987 1 3 SEQ ID NO: 819 ctgaacatcaagaggggca 570 589 SEQ ID NO: 1822 tgcctgcctttgaagtcag 7900 7919 1 3 SEQ ID NO: 820 aacatcaagaggggcatca 573 592 SEQ ID NO: 1823 tgataaaaaccaagatgtt 6290 6309 1 3 SEQ ID NO: 821 acatcaagaggggcatcat 574 593 SEQ ID NO: 1824 atgataaaaaccaagatgt 8289 6308 1 3 SEQ ID NO: 822 tcatttctgccctcctggt 589 608 SEQ ID NO: 1825 accaccagtttgtagatga 7405 7424 1 3 SEQ ID NO: 823 ttcccccagagacagaaga 607 628 SEQ ID NO: 1826 tcttccacatttcaaggaa 10058 10077 1 3 SEQ ID NO: 824 gaagcagccaagcaagtgt 621 640 SEQ ID NO: 1827 acaccttccacattccttc 8071 8090 1 3 SEQ ID NO: 825 ttgtttctggataccgtgt 639 858 SEQ ID NO: 1828 acactaaatacttccacaa 8787 8786 1 3 SEQ ID NO: 826 tgtatggaaactgctccac 655 674 SEQ ID NO: 1829 gtggaggcaacacattaca 2920 2939 1 3 SEQ ID NO: 827 aaactgctccactcacttt 662 681 SEQ ID NO: 1830 aaagaaacagcatttgttt 4532 4551 1 3 SEQ ID NO: 828 actcactttaccgtcaaga 672 691 SEQ ID NO: 1831 tcttacttttccattgagt 10572 10591 1 3 SEQ ID NO: 829 ctttaccgtcaagacgagg 677 696 SEQ ID NO: 1832 cctccagctcctgggaaag 2483 2502 1 3 SEQ ID NO: 830 ttaccgtcaagacgaggaa 679 898 SEQ ID NO: 1833 ttcctaaagctggatgtaa 11169 11188 1 3 SEQ ID NO: 831 acgaggaagggcaatgtgg 600 709 SEQ ID NO: 1834 ccacaagtcatcatctcgt 5956 5975 1 3 SEQ ID NO: 832 cgaggaagggcaatgtggc 691 710 SEQ ID NO: 1835 gccagaagtgagatcctcg 3507 3526 1 3 SEQ ID NO: 833 gaggaagggcaatgtggca 692 711 SEQ ID NO: 1836 tgccagtctccatgacctc 2488 2487 1 3 SEQ ID NO: 834 ggaagggcaatgtggcaac 694 713 SEQ ID NO: 1837 gttgctcttaaggacttcc 13356 13375 1 3 SEQ ID NO: 835 gaagggcaatgtggcaaca 695 714 SEQ ID NO: 1838 tgttgatgaggagtccttc 1801 1820 1 3 SEQ ID NO: 836 caggcatcagcccacttgc 789 788 SEQ ID NO: 1839 gcaagtctttcctggcctg 3011 3030 1 3 SEQ ID NO: 837 aggcatcagcccacttgct 770 789 SEQ ID NO: 1840 agcaagtatttcctggcct 3010 3029 1 3 SEQ ID NO: 838 tcagcccacttgatctcat 775 794 SEQ ID NO: 1841 atgaaagtcaagcatctga 12660 12679 1 3 SEQ ID NO: 839 gtcaactctgatcagcagc 815 834 SEQ ID NO: 1842 gctgactttaaaatctgac 4811 4830 1 3 SEQ ID NO: 840 ggacgctaagaggaagcat 857 878 SEQ ID NO: 1843 atgcactgtttctgagtcc 9331 9350 1 3 SEQ ID NO: 841 aaggagcaacacctcttcc 894 913 SEQ ID NO: 1844 ggaatatcttagcatcctt 13457 13476 1 3 SEQ ID NO: 842 aggagcaacacctcttcat 895 914 SEQ ID NO: 1845 aggaatatcttagcatcct 13456 13475 1 3 SEQ ID NO: 843 caacacctcttcctgcctt 900 919 SEQ ID NO: 1846 aaggctgactctgtggttg 4284 4303 1 3 SEQ ID NO: 844 aacacctcttcctgacttt 901 920 SEQ ID NO: 1847 aaagcaggccgaagctgtt 1087 1088 1 3 SEQ ID NO: 845 acaagaataagtatgggat 925 944 SEQ ID NO: 1848 atccatgatctacatttgt 6786 6805 1 3 SEQ ID NO: 846 caagaataagtatgggatg 926 945 SEQ ID NO: 1849 catcactttacaagccttg 1238 1257 1 3 SEQ ID NO: 847 tagcacaagtgacacagac 948 985 SEQ ID NO: 1850 gtctcttcgttctatgcta 4584 4803 1 3 SEQ ID NO: 848 agcacaagtgacacagact 947 966 SEQ ID NO: 1851 agtctcttcgttctatgct 4583 4802 1 3 SEQ ID NO: 849 gcacaagtgacacagactt 948 987 SEQ ID NO: 1852 aagtgtagtctcctggtgc 5091 5110 1 3 SEQ ID NO: 850 aacttgaagacacaccaaa 970 989 SEQ ID NO: 1853 tttgaggattccatcagtt 7979 7998 1 3 SEQ ID NO: 851 gcttctttggtgaaggtac 1000 1019 SEQ ID NO: 1854 gtacctacttttggcaagc 8364 8383 1 3 SEQ ID NO: 852 ctttggtgaaggtactaag 1004 1023 SEQ ID NO: 1855 cttatgggatttcctaaag 11159 11178 1 3 SEQ ID NO: 853 tactaagaagatgggcctc 1016 1035 SEQ ID NO: 1856 gagggtagtcataacagta 10329 10348 1 3 SEQ ID NO: 854 tttgagagcaccaaatcca 1038 1057 SEQ ID NO: 1857 tggaagtgtcagtggcaaa 10372 10391 1 3 SEQ ID NO: 855 agagcaccaaatccacatc 1042 1061 SEQ ID NO: 1858 gatggatatgaccttctct 4868 4887 1 3 SEQ ID NO: 856 agctgttttgaagactctc 1079 1098 SEQ ID NO: 1859 gagaacatactgggcagct 5872 5891 1 3 SEQ ID NO: 857 tgaaaaaactaaccatctc 1105 1124 SEQ ID NO: 1860 gagaaaatcaatgccttca 7184 7123 1 3 SEQ ID NO: 858 gaaaaaactaaccatctct 1108 1125 SEQ ID NO: 1861 agagccaggtcgagctttc 11044 11063 1 3 SEQ ID NO: 859 tctgagcaaaatatccaga 1122 1141 SEQ ID NO: 1862 tctgatgaggaaactcaga 12252 12271 1 3 SEQ ID NO: 860 tctcttcaataagctggtt 1148 1187 SEQ ID NO: 1863 aacctcccattttttgaga 6318 6337 1 3 SEQ ID NO: 861 ctgagctgagaggcctcag 1168 1187 SEQ ID NO: 1864 ctgatccccgagccctcag 1359 1378 1 3 SEQ ID NO: 862 tgaagcagtcacatctctc 1190 1209 SEQ ID NO: 1865 gagaaaatcaatgccttca 7104 7123 1 3 SEQ ID NO: 863 aagcagtcacatctctctt 1192 1211 SEQ ID NO: 1866 aagaggcagcttctggctt 12289 12308 1 3 SEQ ID NO: 864 ctctcttgccacagctgat 1204 1223 SEQ ID NO: 1867 atcaaaagaagcccaagag 12938 12957 1 3 SEQ ID NO: 865 tcttgccacagctgattga 1207 1226 SEQ ID NO: 1868 tcaaagttaattgggaaga 12271 12280 1 3 SEQ ID NO: 866 cttgccacagctgattgag 1208 1227 SEQ ID NO: 1869 ctcaattttgattttcaag 8520 8539 1 3 SEQ ID NO: 867 tgaggtatccagccccatc 1223 1242 SEQ ID NO: 1870 gatggaaccctctccctca 4725 4744 1 3 SEQ ID NO: 868 tcagtgtggacagcctcag 1259 1278 SEQ ID NO: 1871 ctgacatcttaggcactga 4993 5012 1 3 SEQ ID NO: 869 acatcctccagtggctgaa 1288 1307 SEQ ID NO: 1872 ttcagaagctaagcactgt 7231 7250 1 3 SEQ ID NO: 870 gcacagcagctgcgagaga 1377 1396 SEQ ID NO: 1873 tctctgaaagacaacgtgc 12315 12334 1 3 SEQ ID NO: 871 cagcagctgcgagagatct 1380 1399 SEQ ID NO: 1874 agataacattaaacagctg 13043 13062 1 3 SEQ ID NO: 872 gcgagggatcagcgcagcc 1407 1426 SEQ ID NO: 1875 ggctcaacacagacatcgc 5710 5729 1 3 SEQ ID NO: 873 aagacaaaccctacaggga 1470 1489 SEQ ID NO: 1876 gcaagggttcactgttcct 3928 3947 1 3 SEQ ID NO: 874 caggagctgctggacattg 1491 1510 SEQ ID NO: 1877 caatggagagtccaacctg 4652 4871 1 3 SEQ ID NO: 875 aggagctgctggacattgc 1492 1511 SEQ ID NO: 1878 gcaagggttcactgttcct 7566 7875 1 3 SEQ ID NO: 876 ctgctggacattgctaatt 1497 1516 SEQ ID NO: 1879 aattgggaagaagaggcag 12279 12298 1 3 SEQ ID NO: 877 gattacacctatttgattc 1557 1576 SEQ ID NO: 1880 gaatattttgagaggaatc 8345 8384 1 3 SEQ ID NO: 878 atttgattctgcgggtcat 1567 1588 SEQ ID NO: 1881 atgaagtagaccaacaaat 7153 7172 1 3 SEQ ID NO: 879 tctgcgggtcattggaaat 1574 1593 SEQ ID NO: 1882 atttgtaagaaaatacaga 8428 6447 1 3 SEQ ID NO: 880 aaccatggagcagttaact 1601 1820 SEQ ID NO: 1883 agtttctccatcctaggtt 9954 9973 1 3 SEQ ID NO: 881 ggagcagttaactccagaa 1607 1626 SEQ ID NO: 1884 ttctgaaaatccaatctcc 8392 8411 1 3 SEQ ID NO: 882 cctccagaactcaagtctt 1817 1636 SEQ ID NO: 1885 aagatcgcagactttgagt 11648 11665 1 3 SEQ ID NO: 883 tccagaactcaagtcttca 1819 1638 SEQ ID NO: 1886 tgaactcagaagaattgga 1912 1931 1 3 SEQ ID NO: 884 aagtacaaagccatcactg 1855 1674 SEQ ID NO: 1887 cagtcatgtagaaaaactt 4421 4440 1 3 SEQ ID NO: 885 gccatcactgatgatccag 1664 1683 SEQ ID NO: 1888 ctggaactctctccatggc 10875 10894 1 3 SEQ ID NO: 886 ccatcactgatgatccaga 1665 1684 SEQ ID NO: 1889 tctgaactcagaaggatgg 13991 14010 1 3 SEQ ID NO: 887 atccagaaagctgccatcc 1677 1698 SEQ ID NO: 1890 ggatttcctaaagctggat 11165 11184 1 3 SEQ ID NO: 888 cagaaagctgccatccagg 1680 1699 SEQ ID NO: 1891 cctgaaatacaatgctctg 5510 5529 1 3 SEQ ID NO: 889 acaaggaccaggaggttct 1723 1742 SEQ ID NO: 1892 agaaacagcatttgtttgt 4534 4553 1 3 SEQ ID NO: 890 aggaccaggaggttcttct 1726 1745 SEQ ID NO: 1893 agaagctaagcaatgtcct 7234 7253 1 3 SEQ ID NO: 891 accaggaggttcttcttca 1729 1748 SEQ ID NO: 1894 tgaaggctgactctgtggt 4282 4301 1 3 SEQ ID NO: 892 tcttcagactttccttgat 1742 1761 SEQ ID NO: 1895 atcaggaaggtctcaaaga 2559 2578 1 3 SEQ ID NO: 893 ttcagactttacttgatga 1744 1783 SEQ ID NO: 1896 tcattactcctgggctgaa 11299 11318 1 3 SEQ ID NO: 894 gttgatgaggagtccttca 1802 1821 SEQ ID NO: 1897 tgcatctggctccctcaac 9038 9057 1 3 SEQ ID NO: 895 cttcacaggccgatattaa 1816 1835 SEQ ID NO: 1898 ttaatcgagaggtatgaag 7140 7159 1 3 SEQ ID NO: 896 atattaacaaaattgtcca 1817 1838 SEQ ID NO: 1899 gttaatcgagaggtatgaa 7139 7158 1 3 SEQ ID NO: 897 ggcagatattaacaaaatt 1823 1842 SEQ ID NO: 1900 aattgcattagatgatgcc 6581 6600 1 3 SEQ ID NO: 898 atattaacaaaattgtcca 1828 1847 SEQ ID NO: 1901 tggagtttgtgacaaatat 2752 2771 1 3 SEQ ID NO: 899 acaaaattgtccaaattct 1834 1853 SEQ ID NO: 1902 agaaacagcatttgtttgt 4534 4553 1 3 SEQ ID NO: 900 gagcaagtgaagaactttg 1869 1888 SEQ ID NO: 1903 caaatgacatgatgggctc 5326 5345 1 3 SEQ ID NO: 901 gtgaagaactttgtggctt 1875 1894 SEQ ID NO: 1904 aagcatctgattgactcac 12669 12888 1 3 SEQ ID NO: 902 agaactttgtggcttccca 1879 1898 SEQ ID NO: 1905 tgggcctgccccagattct 8901 8920 1 3 SEQ ID NO: 903 tttgtggcttcccatattg 1884 1903 SEQ ID NO: 1906 caataagatcaatagcaaa 8990 9009 1 3 SEQ ID NO: 904 tggcttcccatattgccaa 1888 1907 SEQ ID NO: 1907 ttggctcacatgaaggcca 7623 7642 1 3 SEQ ID NO: 905 ttcccatattgccaatatc 1892 1911 SEQ ID NO: 1908 gatctacactagggaggaa 12737 12758 1 3 SEQ ID NO: 906 tcccatattgccaatatct 1893 1912 SEQ ID NO: 1909 agatcaaagttaattggga 12268 12287 1 3 SEQ ID NO: 907 ttcccaatatcttgaactc 1900 1919 SEQ ID NO: 1910 gagtcccagtgcccagcaa 9344 9363 1 3 SEQ ID NO: 908 ttggatatccaagatctga 1926 1945 SEQ ID NO: 1911 tcagtataagtacaaccaa 9392 9411 1 3 SEQ ID NO: 909 tccaagatctgaaaaagtt 1933 1952 SEQ ID NO: 1912 aacttccaactgtcatgga 1978 1997 1 3 SEQ ID NO: 910 ctgaaaaagttagtgaaag 1941 1980 SEQ ID NO: 1913 ctttgaagtcagtcttcag 7907 7926 1 3 SEQ ID NO: 911 agttagtgaaagaagttct 1948 1987 SEQ ID NO: 1914 agaatctcaacttccaact 1970 1989 1 3 SEQ ID NO: 912 aatctcaacttccaactgt 1972 1991 SEQ ID NO: 1915 acaggggtcctttatgatt 12342 12381 1 3 SEQ ID NO: 913 gtcatggacttcagaaaat 1989 2008 SEQ ID NO: 1916 atttgaaagaataaatgac 7028 7047 1 3 SEQ ID NO: 914 tcaactctacaaatctgtt 2021 2040 SEQ ID NO: 1917 aacacattgaggctattga 6970 6989 1 3 SEQ ID NO: 915 aactctacaaatctgtttc 2023 2042 SEQ ID NO: 1918 gaaaaaggggattgaagtt 10278 10295 1 3 SEQ ID NO: 916 aaatagaagggaatcttat 2071 2090 SEQ ID NO: 1919 ataagcaaactgttaattt 5449 5468 1 3 SEQ ID NO: 917 agaagggaatcttatattt 2075 2094 SEQ ID NO: 1920 aaatgcactgctgcgttct 4892 4911 1 3 SEQ ID NO: 918 gaagggaatcttatatttg 2076 2095 SEQ ID NO: 1921 caaaaacattttcaacttc 5279 5298 1 3 SEQ ID NO: 919 tgatccaaataactacctt 2093 2112 SEQ ID NO: 1922 aaggaagaaagaaaaatca 3453 3472 1 3 SEQ ID NO: 920 tggatttgcttcagctgac 2150 2169 SEQ ID NO: 1923 gtcagcccagttccttcca 10924 10943 1 3 SEQ ID NO: 921 tttgcttcagctgacctca 2154 2173 SEQ ID NO: 1924 tgaggaaactcagatcaaa 12257 12278 1 3 SEQ ID NO: 922 cttggaaggaaaaggcttt 2183 2202 SEQ ID NO: 1925 aaagcattggtagagcaag 7842 7881 1 3 SEQ ID NO: 923 tggaaggaaaaggctttga 2185 2204 SEQ ID NO: 1926 tcaagtctgtgggattcca 4078 4097 1 3 SEQ ID NO: 924 ggctttgagccaacattgg 2196 2215 SEQ ID NO: 1927 ccaagagatatttaaagcc 12950 12969 1 3 SEQ ID NO: 925 tgagccaacattggaagct 2201 2220 SEQ ID NO: 1928 agctttctgccactgctca 13513 13532 1 3 SEQ ID NO: 926 gagccaacattggaagctc 2202 2221 SEQ ID NO: 1929 gagctttctgccactgctc 13512 13531 1 3 SEQ ID NO: 927 aacattggaagctcttttt 2207 2226 SEQ ID NO: 1930 aaaagaaacagcatttgtt 4531 4550 1 3 SEQ ID NO: 928 tggaagctctttttgggaa 2212 2231 SEQ ID NO: 1931 ttccggcacgtgggttcca 3777 3796 1 3 SEQ ID NO: 929 ctctttttgggaagcaagg 2218 2237 SEQ ID NO: 1932 cattactgactttgcagag 7790 7809 1 3 SEQ ID NO: 930 tttttgggacgcaaggatt 2221 2240 SEQ ID NO: 1933 actcattgaaaaattaaac 6722 6741 1 3 SEQ ID NO: 931 ttttcccagacagtgtcaa 2239 2258 SEQ ID NO: 1934 ttgatgaaatcattgaaaa 6715 6734 1 3 SEQ ID NO: 932 ttggctataccaaagatga 2323 2342 SEQ ID NO: 1935 tcattgctcccggagccaa 2888 2687 1 3 SEQ ID NO: 933 ataccaaagatgataaaca 2329 2348 SEQ ID NO: 1936 tgttgcttttgtaaagtat 8272 8291 1 3 SEQ ID NO: 934 gagaaggatatggtaaatg 2349 2368 SEQ ID NO: 1937 catttcagccttcgggctc 4254 4273 1 3 SEQ ID NO: 935 atggtaaatggaataatgc 2358 2377 SEQ ID NO: 1938 gcatgcctagtttctccat 9948 9965 1 3 SEQ ID NO: 936 tggtaaatggaataatgct 2359 2378 SEQ ID NO: 1939 agcacagtacgaaaaacca 10801 10820 1 3 SEQ ID NO: 937 taaatggaataatgctcag 2362 2381 SEQ ID NO: 1940 ctgaaagagatgaaattta 13059 13078 1 3 SEQ ID NO: 938 tggaataatgctcagtgtt 2368 2385 SEQ ID NO: 1941 aacagatttgaggattcca 7973 7992 1 3 SEQ ID NO: 939 tcagtgttgagaagctgat 2377 2396 SEQ ID NO: 1942 atcacaactcctccactga 9534 9553 1 3 SEQ ID NO: 940 cagtgttgagaagctgatt 2378 2397 SEQ ID NO: 1943 aatcacaactcctccactg 9533 9552 1 3 SEQ ID NO: 941 agtgttgagaagctgatta 2379 2398 SEQ ID NO: 1944 taatcacaactcctccact 9532 9551 1 3 SEQ ID NO: 942 gattaaagatttgaaatcc 2393 2412 SEQ ID NO: 1945 ggatactaagtaccaaatc 6886 6885 1 3 SEQ ID NO: 943 gatttgaaatccaaagaag 2400 2419 SEQ ID NO: 1946 cttccgtttaccagaaatc 8240 8259 1 3 SEQ ID NO: 944 atttgaaatccaaagaagt 2401 2420 SEQ ID NO: 1947 acttccgtttaccagaaat 8239 8258 1 3 SEQ ID NO: 945 atccaaagaagtcccggaa 2408 2427 SEQ ID NO: 1948 ttccaatttccctgtggat 3680 3699 1 3 SEQ ID NO: 946 tccaaagaagtcccggaag 2409 2428 SEQ ID NO: 1949 cttccaatttccctgtgga 3679 3698 1 3 SEQ ID NO: 947 agagcctacctccgcatct 2430 2449 SEQ ID NO: 1950 agattaatccgctggctct 8563 8582 1 3 SEQ ID NO: 948 gagcctacctccgcatctt 2431 2450 SEQ ID NO: 1951 aagattaatccgctggctc 8562 8581 1 3 SEQ ID NO: 949 cttgggagaggagcttggt 2447 2466 SEQ ID NO: 1952 accactgggacctaccaag 12519 12538 1 3 SEQ ID NO: 950 ggagcttggttttgccagt 2456 2475 SEQ ID NO: 1953 actggtggcaaaaccctcc 2726 2745 1 3 SEQ ID NO: 951 ttggttttgccagtctcca 2461 2480 SEQ ID NO: 1954 tggagaagccacactccaa 10763 10782 1 3 SEQ ID NO: 952 cagtctccatgacctccag 2471 2490 SEQ ID NO: 1955 ctggtcgcctgccaaactg 3530 3549 1 3 SEQ ID NO: 953 ctccatgacctccagctcc 2475 2494 SEQ ID NO: 1956 ggagtcattgctcccggag 2864 2683 1 3 SEQ ID NO: 954 ctgggaaagctgcttctga 2493 2512 SEQ ID NO: 1957 tcagaaagctaccttccag 7931 7950 1 3 SEQ ID NO: 955 gaggtcatcaggaagggct 2553 2572 SEQ ID NO: 1958 agccagaagtgagatcctc 3506 3525 1 3 SEQ ID NO: 956 aagaatgacttttttcttc 2574 2593 SEQ ID NO: 1959 gaaggcatctgggagtctt 3827 3848 1 3 SEQ ID NO: 957 cttttttcttcactacatc 2582 2601 SEQ ID NO: 1960 gatgcttacaacactaaag 6099 6118 1 3 SEQ ID NO: 958 catcttcatggagaatgcc 2597 2616 SEQ ID NO: 1961 ggcacttccaaaattgatg 10710 10729 1 3 SEQ ID NO: 959 cttcatggagaatgccttt 2800 2619 SEQ ID NO: 1962 aaagttaattgggaagaag 12273 12292 1 3 SEQ ID NO: 960 aatgcctttgaactcccca 2610 2629 SEQ ID NO: 1963 tgggctggcttcagccatt 5729 5748 1 3 SEQ ID NO: 961 gcctttgaactccccactg 2613 2632 SEQ ID NO: 1964 cagtctgaacattgcaggc 6375 5394 1 3 SEQ ID NO: 962 caaggctggagtaaaactg 2684 2703 SEQ ID NO: 1965 cagtgcaacgaccaacttg 5072 5091 1 3 SEQ ID NO: 963 tggagtaaaactggaagta 2690 2709 SEQ ID NO: 1966 tactccaacgccagctcca 3051 3070 1 3 SEQ ID NO: 964 ggaagtagccaacatgcag 2702 2721 SEQ ID NO: 1967 ctgccatctcgagagttcc 4098 4117 1 3 SEQ ID NO: 965 tttgtgacaaatatgggca 2757 2776 SEQ ID NO: 1968 tgcctttgtgtacaccaaa 11228 11247 1 3 SEQ ID NO: 966 tgtgacaaatatgggcatc 2759 2778 SEQ ID NO: 1969 gatgggtctctacgccaca 4377 4398 1 3 SEQ ID NO: 967 ggacttcgctaggagtggg 2786 2805 SEQ ID NO: 1970 cccaaggccacaggggtcc 12333 12352 1 3 SEQ ID NO: 968 gtggggtccagatgaacac 2800 2819 SEQ ID NO: 1971 gtgttctagacctctccac 4171 4190 1 3 SEQ ID NO: 969 ttccacgagtcgagtctgg 2828 2845 SEQ ID NO: 1972 ccagaatctgtaccaggaa 12554 12573 1 3 SEQ ID NO: 970 agtcgggtctggaggctca 2833 2852 SEQ ID NO: 1973 tgagaactacgagctgact 4799 4818 1 3 SEQ ID NO: 971 tcgggtctggaggctcatg 2835 2854 SEQ ID NO: 1974 catgaaggccaaattccga 7831 7650 1 3 SEQ ID NO: 972 aaaagctgggaagctgaag 2881 2880 SEQ ID NO: 1975 cttccagacacctgatttt 7943 7982 1 3 SEQ ID NO: 973 aagctgaagtttatcattc 2871 2890 SEQ ID NO: 1976 gaatttacaattgttgctt 6261 6280 1 3 SEQ ID NO: 974 gagaccagtcaagctgctc 2900 2919 SEQ ID NO: 1977 gagcttcaggaagcttctc 13206 13225 1 3 SEQ ID NO: 975 gcaacacattacatttggt 2926 2945 SEQ ID NO: 1978 accagtcagatattgttgc 10183 10202 1 3 SEQ ID NO: 976 acattacatttggtctcta 2931 2950 SEQ ID NO: 1979 tagaatatgaactaaatgt 11881 11900 1 3 SEQ ID NO: 977 cattacatttggtctctac 2932 2951 SEQ ID NO: 1980 gtagctgagaaaatcaatg 7098 7117 1 3 SEQ ID NO: 978 aaacggaggtgatcccacc 2956 2975 SEQ ID NO: 1981 ggtggataccctgaagttt 3197 3216 1 3 SEQ ID NO: 979 attgagaacaggcagtcct 2979 2998 SEQ ID NO: 1982 aggaaaagcgcacctcaat 12023 12042 1 3 SEQ ID NO: 980 tgagaacaggcagtcctgg 2981 3000 SEQ ID NO: 1983 ccagcttccccacatctca 8333 8352 1 3 SEQ ID NO: 981 ctgcacctcaggcgcttac 3035 3054 SEQ ID NO: 1984 gtaagaaaatacagagcag 8432 6451 1 3 SEQ ID NO: 982 tccacagactccgcctcct 3066 3085 SEQ ID NO: 1985 aggacagagccttggtgga 3184 3203 1 3 SEQ ID NO: 983 ctgaccggggacaccagat 3093 3112 SEQ ID NO: 1986 atctgatgaggaaactcag 12251 12270 1 3 SEQ ID NO: 984 tagagctggaactgaggcc 3112 3131 SEQ ID NO: 1987 ggcctctctggggcatcta 5138 5155 1 3 SEQ ID NO: 985 ctatgagctccagagagag 3167 3188 SEQ ID NO: 1988 ctctcacaaaaaagtatag 8541 6560 1 3 SEQ ID NO: 986 cttggtggataccctgaag 3194 3213 SEQ ID NO: 1989 cttcaggaagcttctcaag 13209 13228 1 3 SEQ ID NO: 987 ttgtaactcaagcagaagg 3214 3233 SEQ ID NO: 1990 ccttacacaataatcacaa 9522 9641 1 3 SEQ ID NO: 988 taactcaagcagaaggtgc 3217 3236 SEQ ID NO: 1991 gcacctagctggaaagtta 6947 8966 1 3 SEQ ID NO: 989 gcagaaggtgcgaagcaga 3225 3244 SEQ ID NO: 1992 tctgtgggattccatctgc 4083 4102 1 3 SEQ ID NO: 990 cagaaggtgcgaagcagac 3228 3245 SEQ ID NO: 1993 gtctgtgggattccatctg 4082 4101 1 3 SEQ ID NO: 991 gtatgaccttgtccagtga 3280 3299 SEQ ID NO: 1994 tcaccaacggagaacatac 10843 10862 1 3 SEQ ID NO: 992 tatgaccttgtccagtgaa 3281 3300 SEQ ID NO: 1995 ttcaccaacggagaacata 10842 10881 1 3 SEQ ID NO: 993 gaagtccaaattccggatt 3297 3316 SEQ ID NO: 1996 aatctcaagctttctcttc 10044 10083 1 3 SEQ ID NO: 994 gagggcaaaacgtcttaca 3363 3382 SEQ ID NO: 1997 tgtacaactggtccgcctc 4207 4226 1 3 SEQ ID NO: 995 agggcaaaacgtcttacag 3364 3383 SEQ ID NO: 1998 ctgttaggacaccagccct 4054 4073 1 3 SEQ ID NO: 996 gactcaccctggacattca 3382 3401 SEQ ID NO: 1999 tgaaattcaatcacaagtc 9068 9087 1 3 SEQ ID NO: 997 ctggacattcagaacaaga 3390 3409 SEQ ID NO: 2000 tcttttcttttcagcccag 9218 9237 1 3 SEQ ID NO: 998 tcatgggcgacctaagttg 3427 3446 SEQ ID NO: 2001 caactgcagacatatatga 6827 6848 1 3 SEQ ID NO: 999 tgggcgacctaagttgtga 3430 3449 SEQ ID NO: 2002 tcactccattaacctccca 6308 8327 1 3 SEQ ID NO: 1000 agttgtgacacaaaggaag 3441 3480 SEQ ID NO: 2003 cttcttttccaattgaact 13830 13849 1 3 SEQ ID NO: 1001 tgacacaaaggaagaaaga 3448 3465 SEQ ID NO: 2004 tcttcatcttcatctgtca 10212 10231 1 3 SEQ ID NO: 1002 gacacaaaggaagaaagaa 3447 3466 SEQ ID NO: 2005 ttcttcatcttcatctgtc 10211 10230 1 3 SEQ ID NO: 1003 ggaagaaagaaaaatcaag 3455 3474 SEQ ID NO: 2006 cttgtcatgcctacgttcc 11340 11359 1 3 SEQ ID NO: 2007 aaaaagcgatggccgggtc 3947 3988 SEQ ID NO: 2313 gaccttgcaagaatatttt 6335 6354 1 3 SEQ ID NO: 2008 gtcaaatataccttgaaca 3963 3982 SEQ ID NO: 2314 tgttaacaaattccttgac 7355 7374 1 3 SEQ ID NO: 2009 tgaacaagaacagtttgaa 3978 3995 SEQ ID NO: 2315 ttcaagttcctgaccttca 8302 8321 1 3 SEQ ID NO: 2010 agtttgaaaattgagattc 3987 4008 SEQ ID NO: 2316 gaatctggctccctcaact 9039 9058 1 3 SEQ ID NO: 2011 gtttgaaaattgagattcc 3988 4007 SEQ ID NO: 2317 ggaaataccaagtcaaaac 10446 10485 1 3 SEQ ID NO: 2012 ttgaaaattgagattcctt 3990 4009 SEQ ID NO: 2318 aaggaaaagcgcacctcaa 12022 12041 1 3 SEQ ID NO: 2013 ctaaagatgttagagactg 4038 4057 SEQ ID NO: 2319 cagttgaccacaagcttag 10537 10556 1 3 SEQ ID NO: 2014 atgttagagactgttagga 4044 4063 SEQ ID NO: 2320 tccttaacaccttccacat 8065 8084 1 3 SEQ ID NO: 2015 cagccatccacttcaagtc 4066 4085 SEQ ID NO: 2321 gacttctctagtcaggctg 8805 8824 1 3 SEQ ID NO: 2016 agccctccacttcaagtat 4067 4086 SEQ ID NO: 2322 agacatcgctgggctggct 5720 5739 1 3 SEQ ID NO: 2017 ccatctgccatctcgagag 4094 4113 SEQ ID NO: 2323 ctctcaaatgacatgatgg 5322 5341 1 3 SEQ ID NO: 2018 attcccaagttgtatcaac 4134 4153 SEQ ID NO: 2324 gttgagaagccccaagaat 6246 5265 1 3 SEQ ID NO: 2019 tcaactgcaagtgcctctc 4148 4187 SEQ ID NO: 2325 gagatcaagacactgttga 8835 8854 1 3 SEQ ID NO: 2020 agtattctagacctctcca 4170 4189 SEQ ID NO: 2326 tggaaccctctccctcacc 4727 4746 1 3 SEQ ID NO: 2021 ctccacgaatgtctacagc 4184 4203 SEQ ID NO: 2327 gctggtaacctaaaaggag 5580 5599 1 3 SEQ ID NO: 2022 cacgaatgtctacagcaac 4187 4206 SEQ ID NO: 2328 gttgcccaccatcatcgtg 11663 11662 1 3 SEQ ID NO: 2023 acgaatgtctacagcaact 4188 4207 SEQ ID NO: 2329 agttgcccaccatcatcgt 11662 11681 1 3 SEQ ID NO: 2024 tcctacagtgatggcaacc 4224 4243 SEQ ID NO: 2330 agttagttgctcttaagga 13351 13370 1 3 SEQ ID NO: 2025 cgttaccacatgaaggctg 4272 4291 SEQ ID NO: 2331 cagcaagtacctgagaacg 8603 8822 1 3 SEQ ID NO: 2026 gaaggctgactctgtggtt 4283 4302 SEQ ID NO: 2332 ttcaaaacgagcttcagga 13161 13180 1 3 SEQ ID NO: 2027 tgtggttgacctgctttcc 4295 4314 SEQ ID NO: 2333 ggaaagttaaaaccacaca 6957 6976 1 3 SEQ ID NO: 2028 cctgctttcctacaatgtg 4304 4323 SEQ ID NO: 2334 cacaccttgacattgcagg 11080 11099 1 3 SEQ ID NO: 2029 ctgctttcctacaatgtgc 4305 4324 SEQ ID NO: 2335 gcacaccttgacattgcag 11079 11096 1 3 SEQ ID NO: 2030 tcctacaatgtgcaaggat 4311 4330 SEQ ID NO: 2336 atccgctggctctgaagga 8569 8588 1 3 SEQ ID NO: 2031 tatgaccacaagaatacgt 4344 4363 SEQ ID NO: 2337 acgtccgtgtgccttcata 9976 9995 1 3 SEQ ID NO: 2032 atgaccacaagaatacgtc 4345 4364 SEQ ID NO: 2338 gacgtccgtgtgccttcat 9975 9994 1 3 SEQ ID NO: 2033 gaatacgtctacactatca 4355 4374 SEQ ID NO: 2339 tgattatctgaattcattc 6479 6498 1 3 SEQ ID NO: 2034 tttctagattcgaatatca 4398 4417 SEQ ID NO: 2340 tgatttacatgatttgaaa 6677 6696 1 3 SEQ ID NO: 2035 gattagaatatcaaattca 4404 4423 SEQ ID NO: 2341 tgaagtagctgagaaaatc 7094 7113 1 3 SEQ ID NO: 2036 gaaccaacccagtctcaaa 4441 4460 SEQ ID NO: 2342 tttgaaaaattctcttttc 9206 9225 1 3 SEQ ID NO: 2037 cccagtctcaaaaggttta 4448 4467 SEQ ID NO: 2343 taaattcattactcctggg 11294 11313 1 3 SEQ ID NO: 2038 ctcaaaaggtttactaata 4454 4473 SEQ ID NO: 2344 tattcaaaactgagttgag 12223 12242 1 3 SEQ ID NO: 2039 tcaaaaggtttactaatat 4455 4474 SEQ ID NO: 2345 atattcaaaactgagttga 12222 12241 1 3 SEQ ID NO: 2040 aaaaggtttactaatattc 4457 4476 SEQ ID NO: 2346 gaatttgaaagttcgtttt 9272 9291 1 3 SEQ ID NO: 2041 gaaacagcatttgtttgtc 4535 4554 SEQ ID NO: 2347 gacagcatcttcgtgtttc 11206 11225 1 3 SEQ ID NO: 2042 atttgtttgtcaaagaagt 4543 4562 SEQ ID NO: 2348 acttaaaaaatataaaaat 8014 8033 1 3 SEQ ID NO: 2043 tcaagattgatgggcagtt 4581 4580 SEQ ID NO: 2349 aactctcaagtcaagttga 13414 13433 1 3 SEQ ID NO: 2044 ttcagagtctcttcgttct 4578 4597 SEQ ID NO: 2350 agaagatggcaaatttgaa 11987 12006 1 3 SEQ ID NO: 2045 cagagtctattcgttctat 4580 4599 SEQ ID NO: 2351 atagcatggacttcttctg 8865 8884 1 3 SEQ ID NO: 2046 atgctaaaggcacatatgg 4597 4616 SEQ ID NO: 2352 ccatttgagatcacggcat 9237 9256 1 3 SEQ ID NO: 2047 gcacatatggcctgtcttg 4606 4625 SEQ ID NO: 2353 caagttggcaagtaagtgc 9364 9383 1 3 SEQ ID NO: 2048 gagtccaacctgaggttta 4659 4676 SEQ ID NO: 2354 taaagtgccacttttactc 6182 6201 1 3 SEQ ID NO: 2049 agtccaacctgaggtttaa 4860 4679 SEQ ID NO: 2355 ttaacagggaagatagact 9300 9319 1 3 SEQ ID NO: 2050 cctacctccaaggcaccaa 4884 4703 SEQ ID NO: 2356 ttggcaagtaagtgctagg 9368 9387 1 3 SEQ ID NO: 2051 gaagatggaaccctctccc 4722 4741 SEQ ID NO: 2357 gggaagaagaggcagcttc 12283 12302 1 3 SEQ ID NO: 2052 tgatctgcaaagtggcatc 4754 4773 SEQ ID NO: 2358 gatgaggaaactcagatca 12255 12274 1 3 SEQ ID NO: 2053 gatctgcaaagtggcatca 4755 4774 SEQ ID NO: 2359 tgatgaggaaactcagatc 12254 12273 1 3 SEQ ID NO: 2054 gcttccctaaagtatgaga 4785 4804 SEQ ID NO: 2360 tctcgtgtctaggaaaagc 5969 5988 1 3 SEQ ID NO: 2055 gtatgagaactacgagctg 4766 4815 SEQ ID NO: 2361 cagcttaagagacacatac 6912 6931 1 3 SEQ ID NO: 2056 tctaacaagatggatatga 4860 4879 SEQ ID NO: 2362 tcattttccaactaataga 13024 13043 1 3 SEQ ID NO: 2057 ctgctgcgttctgaatatc 4899 4918 SEQ ID NO: 2363 gatacaagaaaaactgcag 6893 6912 1 3 SEQ ID NO: 2058 tcattgaggttcttcagcc 4932 4951 SEQ ID NO: 2364 ggctcatatgctgaaatga 5340 5359 1 3 SEQ ID NO: 2059 ttctggatcactaaattcc 4855 4974 SEQ ID NO: 2365 ggaaggccaaggcccagaa 12541 12560 1 3 SEQ ID NO: 2060 ccatggtcttgagttaaat 4973 4992 SEQ ID NO: 2366 atttttattcctgccatgg 10095 10114 1 3 SEQ ID NO: 2061 tcttaggcactgacaccat 4999 5018 SEQ ID NO: 2367 attttttgcaagttaaaga 14011 14030 1 3 SEQ ID NO: 2062 acaaggcgacactaaggat 5032 5051 SEQ ID NO: 2368 atccatgatctacatttgt 6786 6805 1 3 SEQ ID NO: 2063 tgcaacgaccaacttgaag 5075 5094 SEQ ID NO: 2369 cttcagggaacacaatgca 5177 5196 1 3 SEQ ID NO: 2064 caacttgaagtgtagtctc 5084 5103 SEQ ID NO: 2370 gagatgagagatgccgttg 6231 6250 1 3 SEQ ID NO: 2065 gctggagaatgagctgaat 5108 5127 SEQ ID NO: 2371 attctcttttcttttcagc 9214 9233 1 3 SEQ ID NO: 2066 gcagaacttggcctctctg 5127 5146 SEQ ID NO: 2372 cagatacaagaaaaactgc 6891 6910 1 3 SEQ ID NO: 2067 tctctgaggcatctatgaa 5140 5159 SEQ ID NO: 2373 ttcattcaattgggagaga 6491 6510 1 3 SEQ ID NO: 2068 tctggggcatctatgaaat 5142 5161 SEQ ID NO: 2374 atttgtaagaaaatacaga 6428 6447 1 3 SEQ ID NO: 2069 aacacaatgccaaattcag 5185 5204 SEQ ID NO: 2375 ctgaagcattaaaactgtt 7498 7517 1 3 SEQ ID NO: 2070 ctcacagagctatcactgg 5223 5242 SEQ ID NO: 2376 ccagatgctgaacagtgag 6141 8160 1 3 SEQ ID NO: 2071 tgagaagtgcttatcaggc 5239 5258 SEQ ID NO: 2377 gcctacgttccatgtccca 11348 11367 1 3 SEQ ID NO: 2072 ttcaaggtcagtcaagaag 5295 5314 SEQ ID NO: 2378 cttcagtgcagaatatgaa 11969 11988 1 3 SEQ ID NO: 2073 aatgacatgatgggctcat 5328 5347 SEQ ID NO: 2379 atgattatctgaattcatt 6478 6497 1 3 SEQ ID NO: 2074 gctcatatgctgaaatgaa 5341 5380 SEQ ID NO: 2380 ttcagccattgacatgagc 5738 5757 1 3 SEQ ID NO: 2075 atatgctgaaatgaaattt 5345 5364 SEQ ID NO: 2381 aaatagctattgctaatat 6694 6713 1 3 SEQ ID NO: 2076 tctgaacattgcaggctta 5378 5397 SEQ ID NO: 2382 taagaaccagaagatcaga 10988 11007 1 3 SEQ ID NO: 2077 gaacattgcaggcttatca 5381 5400 SEQ ID NO: 2383 tgatatcgacgtgaggttc 12482 12501 1 3 SEQ ID NO: 2078 tgcaggcttatcactggac 5387 5406 SEQ ID NO: 2384 gtcctggattccacatgca 11844 11863 1 3 SEQ ID NO: 2079 tcaaaacttgacaacattt 5412 5431 SEQ ID NO: 2385 aacttccttgacatgttga 7362 7381 1 3 SEQ ID NO: 2080 atttacagctctgacaagt 5427 5446 SEQ ID NO: 2386 acttaaaaaatataaaaat 8014 8033 1 3 SEQ ID NO: 2081 ctctgacaagttttataag 5435 5454 SEQ ID NO: 2387 cttacttgaattccaagag 10686 10685 1 3 SEQ ID NO: 2082 gttaatttacagctacagc 5480 5479 SEQ ID NO: 2388 gctgcatgtggctggtaac 5570 5589 1 3 SEQ ID NO: 2083 ttctctggtaactacttta 5483 5502 SEQ ID NO: 2389 taaaagattactttgagaa 7267 7288 1 3 SEQ ID NO: 2084 cctaaaaggagcctaccaa 5588 5607 SEQ ID NO: 2390 ttggcaagtaagtgctagg 9368 9387 1 3 SEQ ID NO: 2085 aaaaggagcctacccaaat 5591 5610 SEQ ID NO: 2391 atttacaattgttgctttt 6263 6282 1 3 SEQ ID NO: 2086 aggagcctaccaaaataat 5594 5613 SEQ ID NO: 2392 attacctatgatttctcct 10119 10138 1 3 SEQ ID NO: 2087 ataatgaaataaaacacat 5608 5627 SEQ ID NO: 2393 atgtcaaacactttgttat 7057 7076 1 3 SEQ ID NO: 2088 aaaacacatctatgccatc 5618 5637 SEQ ID NO: 2394 gatgaagatgacgactttt 12150 12169 1 3 SEQ ID NO: 2089 tgctaaggttcagggtgtg 5678 5697 SEQ ID NO: 2395 cacaagtcgattcccagca 9079 9098 1 3 SEQ ID NO: 2090 gagtttagccatcggctca 5697 5716 SEQ ID NO: 2396 tgaggtgactcagagactc 7442 7461 1 3 SEQ ID NO: 2091 gctggcttcagccattgac 5732 5751 SEQ ID NO: 2397 gtcagtgaagttctccagc 8588 8607 1 3 SEQ ID NO: 2092 atttcagcaatgtcttccg 5782 5801 SEQ ID NO: 2398 cggagcatgggagtgaaat 8620 8639 1 3 SEQ ID NO: 2093 tttcagcaatgtattccgt 5783 5802 SEQ ID NO: 2399 acggagcatgggagtgaaa 8619 8838 1 3 SEQ ID NO: 2094 ttcagcaatgtcttccgtt 5784 5803 SEQ ID NO: 2400 aacggagcatgggagtgaa 8618 8637 1 3 SEQ ID NO: 2095 cagcaatgtcttccgttct 5786 5805 SEQ ID NO: 2401 agaagtgtcttcaaagctg 12404 12423 1 3 SEQ ID NO: 2096 tgtcttccgttctgtaatg 5792 5811 SEQ ID NO: 2402 cattcaattgggagagaca 6493 6512 1 3 SEQ ID NO: 2097 gtcttccgttctgtaatgg 5793 5812 SEQ ID NO: 2403 ccattcagtctctcaagac 12967 12986 1 3 SEQ ID NO: 2098 atgggaaactcgctctctg 5851 5870 SEQ ID NO: 2404 cagataaaaaactcaccat 12205 12224 1 3 SEQ ID NO: 2099 ggagcacatactgggcagc 5871 5890 SEQ ID NO: 2405 gctgttttgaagactctcc 1080 1099 1 3 SEQ ID NO: 2100 gttgaaagcagaacctctg 5906 5925 SEQ ID NO: 2406 cagaattcataatcccaac 8268 8285 1 3 SEQ ID NO: 2101 gtctaggaaaagcatcagt 5975 6994 SEQ ID NO: 2407 actgcaagatttttcagac 13604 13623 1 3 SEQ ID NO: 2102 agcatcagtgcagctcttg 5985 6004 SEQ ID NO: 2408 caagaacctgttagttgct 13343 13362 1 3 SEQ ID NO: 2103 ttgaacacaaagtcagtgc 6001 6020 SEQ ID NO: 2409 gcacatcaatattgatcaa 6410 6429 1 3 SEQ ID NO: 2104 gcagacaggcacctggaaa 6036 6057 SEQ ID NO: 2410 tttcagatggcattgctgc 11602 11621 1 3 SEQ ID NO: 2105 gaaactccagacccaattt 6053 6072 SEQ ID NO: 2411 aaatcccatccaggttttc 8029 8048 1 3 SEQ ID NO: 2106 acaatgaatacagccagga 6076 6095 SEQ ID NO: 2412 tcctttggctgtgctttgt 9674 9693 1 3 SEQ ID NO: 2107 cttggatgcttacaacact 6095 6114 SEQ ID NO: 2413 agtgaagttctccagcaag 8591 8610 1 3 SEQ ID NO: 2108 ttggcgtggagcttactgg 6124 6143 SEQ ID NO: 2414 ccagaattcataatcccaa 8265 8284 1 3 SEQ ID NO: 2109 cacttttactcagtgagcc 6190 6209 SEQ ID NO: 2415 ggctattgatgttagagtg 6980 6999 1 3 SEQ ID NO: 2110 tttagagatgagagatgcc 6227 6246 SEQ ID NO: 2416 ggcatgatgctcatttaaa 9169 9188 1 3 SEQ ID NO: 2111 gagaagccccaagaattta 6249 6286 SEQ ID NO: 2417 taaagccattcagtctctc 12962 12981 1 3 SEQ ID NO: 2112 caattgttgcttttgtaaa 6268 6287 SEQ ID NO: 2418 tttaaccagtcagatattg 10179 10198 1 3 SEQ ID NO: 2113 ttttgtaaagtatgataaa 6278 6297 SEQ ID NO: 2419 tttattgctgaatccaaaa 13647 13666 1 3 SEQ ID NO: 2114 ttgtaaagtctgataaaaa 6280 6299 SEQ ID NO: 2420 ttttgagaggaatcgacaa 6350 6369 1 3 SEQ ID NO: 2115 ttcactccattaacctccc 6307 6326 SEQ ID NO: 2421 gggaaaaaacaggcttgaa 9568 9587 1 3 SEQ ID NO: 2116 ttttgagaccttgcaagaa 6329 6348 SEQ ID NO: 2422 ttctctctatgggaaaaaa 9558 9577 1 3 SEQ ID NO: 2117 accttgcaagaatattttg 6336 6355 SEQ ID NO: 2423 caaaagaagcccaagaggt 12940 12959 1 3 SEQ ID NO: 2118 tcaatattgatcaatttgt 6415 6434 SEQ ID NO: 2424 acaaagcagattatgttga 11821 11840 1 3 SEQ ID NO: 2119 cagagcagccctgggaaaa 6443 6462 SEQ ID NO: 2425 ttttcagaccaactctctg 13614 13633 1 3 SEQ ID NO: 2120 cctgggaaaactcccacag 6452 6471 SEQ ID NO: 2426 ctgtctctggtcagccagg 7716 7735 1 3 SEQ ID NO: 2121 actcccacagcaagctaat 6461 6460 SEQ ID NO: 2427 attacacttcctttcgagt 12861 12880 1 3 SEQ ID NO: 2122 aattcattcaatttttaga 6489 6508 SEQ ID NO: 2428 tctcttcctccatggaatt 10471 10490 1 3 SEQ ID NO: 2123 ttcaattgggagagacaag 6495 6514 SEQ ID NO: 2429 cttggagtgccagtttgaa 11800 11819 1 3 SEQ ID NO: 2124 aggagaaactgactgctct 6526 6545 SEQ ID NO: 2430 agagcttatgggatttcct 11155 11174 1 3 SEQ ID NO: 2125 actgactgctctcacacaa 6533 6552 SEQ ID NO: 2431 ttttggcaagctatacagt 8372 8391 1 3 SEQ ID NO: 2126 gactgctctcacaaaaaag 6536 6555 SEQ ID NO: 2432 ctttgtgagtttatcagtc 9687 9706 1 3 SEQ ID NO: 2127 cagacatatatgatacaat 6633 6652 SEQ ID NO: 2433 attggatatccaagatctg 1925 1944 1 3 SEQ ID NO: 2128 aatttgatcagtatattaa 6649 6668 SEQ ID NO: 2434 ttaaaagaaatcttcaatt 13807 13826 1 3 SEQ ID NO: 2129 tatgatttacatgatttga 6675 5716 SEQ ID NO: 2435 tcaatgattatatcccata 13120 13139 1 3 SEQ ID NO: 2130 tttgaaaatagctattgct 6689 5751 SEQ ID NO: 2436 agcacagaaaaaattcaaa 13856 13875 1 3 SEQ ID NO: 2131 ttgaaaatagctattgcta 6690 5801 SEQ ID NO: 2437 tagcacagaaaaaattcaa 13855 13874 1 3 SEQ ID NO: 2132 aatagctattgctaatatt 6695 5802 SEQ ID NO: 2438 aataaatggagtctttatt 14076 14095 1 3 SEQ ID NO: 2133 attattgatgaaatcattg 6711 5803 SEQ ID NO: 2439 caataccagaattcataat 8260 8279 1 3 SEQ ID NO: 2134 aaagtcttgatgagcacta 6739 5805 SEQ ID NO: 2440 tagtgattacacttccttt 12856 12875 1 3 SEQ ID NO: 2135 aagtcttgatgagcactat 6740 5811 SEQ ID NO: 2441 atagcaacactaaatactt 6761 8780 1 3 SEQ ID NO: 2136 ttgatgahcactatcatat 6745 5812 SEQ ID NO: 2442 atatccaagatgagatcaa 13093 13112 1 3 SEQ ID NO: 2137 taattttagtaaaaacaat 6769 5870 SEQ ID NO: 2443 attgagattccctccatta 11694 11713 1 3 SEQ ID NO: 2138 ttttagtaaaaacaatcca 6772 5890 SEQ ID NO: 2444 tggagtgccagtttgaaaa 11802 11821 1 3 SEQ ID NO: 2139 acatttgtttattgaaaat 6797 5925 SEQ ID NO: 2445 atttcctaaagctggatgt 11167 11186 1 3 SEQ ID NO: 2140 attgattttaacaaaagtg 6816 6994 SEQ ID NO: 2446 cactgttccagttgtcaat 9863 9882 1 3 SEQ ID NO: 2141 attttaacaaaagtggaag 6820 6004 SEQ ID NO: 2447 cttcaaagacttaaaaaat 8006 8025 1 3 SEQ ID NO: 2142 aaatcagaatccagataca 6880 6020 SEQ ID NO: 2448 tgtaccataagccatattt 10080 10099 1 3 SEQ ID NO: 2143 gaatccagatacaagaaaa 6886 6057 SEQ ID NO: 2449 ttttctaaacttgaaattc 9057 9076 1 3 SEQ ID NO: 2144 ttaagagacacatacagaa 6916 6072 SEQ ID NO: 2450 ttcttaaacattcctttaa 9483 9502 1 3 SEQ ID NO: 2145 atccagcacctagctggaa 6942 6095 SEQ ID NO: 2451 ttccaatttccctgtggat 3660 3699 1 3 SEQ ID NO: 2146 tgagcatgtcaaacacttt 7052 6114 SEQ ID NO: 2452 aaagtgccacttttactca 6183 6202 1 3 SEQ ID NO: 2147 gagcatgtcaaacactttg 7053 6143 SEQ ID NO: 2453 caaatgacatgatgggctc 5326 5345 1 3 SEQ ID NO: 2148 aaacactttgttataaatc 7062 6209 SEQ ID NO: 2454 gattatatcccatatgttt 13125 13144 1 3 SEQ ID NO: 2149 tgagaaaatcaatgccttc 7103 6246 SEQ ID NO: 2455 gaaggaaaagcgcacctca 12021 12040 1 3 SEQ ID NO: 2150 tatgaagtagaccaacaaa 7152 6286 SEQ ID NO: 2456 tttgtggagggtagtcata 10323 10342 1 3 SEQ ID NO: 2151 aagtagaccaacaaatcca 7156 6287 SEQ ID NO: 2457 tggatgaagatgacgactt 12148 12167 1 3 SEQ ID NO: 2152 aagttgaaggagactattc 7215 6297 SEQ ID NO: 2458 gaataccaatgctgaactt 10160 10179 1 3 SEQ ID NO: 2153 acaagttaagataaaagat 7256 7275 SEQ ID NO: 2459 atctaaattcagttcttgt 11326 11345 1 3 SEQ ID NO: 2154 aagataaaagattactttg 7263 7282 SEQ ID NO: 2460 caaaatagaagggaatctt 2069 2088 1 3 SEQ ID NO: 2155 gattactttgagaaattag 7272 7291 SEQ ID NO: 2461 ctaaacttgaaattcaatc 9061 9080 1 3 SEQ ID NO: 2156 tgagaaattagttggattt 7280 7299 SEQ ID NO: 2462 aaatccgtgaggtgactca 7435 7454 1 3 SEQ ID NO: 2157 aaattagttggatttattg 7284 7303 SEQ ID NO: 2463 caattttgagaatgaattt 10411 10430 1 3 SEQ ID NO: 2158 tggatttattgatgatgct 7292 7311 SEQ ID NO: 2464 agcatgcctagtttctcca 9945 9964 1 3 SEQ ID NO: 2159 tcattgaagatgttaacaa 7345 7364 SEQ ID NO: 2465 ttgtagatgaaaccaatga 7414 7433 1 3 SEQ ID NO: 2160 cattgaagatgttaacaaa 7346 7365 SEQ ID NO: 2466 tttgtagatgaaaccaatg 7413 7432 1 3 SEQ ID NO: 2161 attgaagatgttaacaaat 7347 7366 SEQ ID NO: 2467 atttaagtatgatttcaat 10487 10506 1 3 SEQ ID NO: 2162 ttgaagatgttaacaaatt 7348 7367 SEQ ID NO: 2468 aatttaagtatgatttcaa 10486 10505 1 3 SEQ ID NO: 2163 tgaagatgttaacaaattc 7349 7368 SEQ ID NO: 2469 gaatttaagtatgatttca 10485 10504 1 3 SEQ ID NO: 2164 acatgttgataaagaaatt 7372 7391 SEQ ID NO: 2470 aattccctgaagttgatgt 11479 11498 1 3 SEQ ID NO: 2165 tttgattaccaccagtttg 7398 7417 SEQ ID NO: 2471 caaattgaacatccccaaa 8783 8802 1 3 SEQ ID NO: 2166 caaaatccgtgaggtgact 7433 7452 SEQ ID NO: 2472 agtccccctaacagatttg 7964 7983 1 3 SEQ ID NO: 2167 aaaatccgtgaggtgactc 7434 7453 SEQ ID NO: 2473 gagtgaaatgctgtttttt 8630 8649 1 3 SEQ ID NO: 2168 aggtgactcagagactcaa 7444 7463 SEQ ID NO: 2474 ttgatgatatctggaacct 10723 10742 1 3 SEQ ID NO: 2169 gtgaaattcaggctctgga 7465 7484 SEQ ID NO: 2475 tccaatctcctcttttcac 8401 8420 1 3 SEQ ID NO: 2170 gttgcagtgtatctggaaa 7539 7558 SEQ ID NO: 2476 tttcaagcaaatgcacaac 8532 8551 1 3 SEQ ID NO: 2171 ttaagttcagcatctttgg 7608 7627 SEQ ID NO: 2477 ccaatgctgaactttttaa 10165 10184 1 3 SEQ ID NO: 2172 tgaaggccaaattccgaga 7633 7652 SEQ ID NO: 2478 tctcctttcttcatcttca 10205 10224 1 3 SEQ ID NO: 2173 aatgtatcaaatggacatt 7676 7695 SEQ ID NO: 2479 aatgaagtccggattcatt 11013 11032 1 3 SEQ ID NO: 2174 attcagcaggaacttcaac 7692 7711 SEQ ID NO: 2480 gttgagaagccccaagaat 6246 6265 1 3 SEQ ID NO: 2175 acctgtctctggtcagcca 7714 7733 SEQ ID NO: 2481 tggcaagtaagtgctaggt 9369 9388 1 3 SEQ ID NO: 2176 cctgtctctggtcagccag 7715 7734 SEQ ID NO: 2482 ctggacttctctagtcagg 8802 8821 1 3 SEQ ID NO: 2177 ggtcagccaggtttatagc 7724 7743 SEQ ID NO: 2483 gctaaaggagcagttgacc 10527 10546 1 3 SEQ ID NO: 2178 ccaggtttatagcacactt 7730 7748 SEQ ID NO: 2484 aagtccggattcattctgg 11017 11036 1 3 SEQ ID NO: 2179 gtttatagcacacttgtca 7734 7753 SEQ ID NO: 2485 tgacctgtccattcaaaac 13873 13692 1 3 SEQ ID NO: 2180 acttgtcacctacctttct 7745 7764 SEQ ID NO: 2486 agaaaaaggggattgaagt 10275 10294 1 3 SEQ ID NO: 2181 ctgattggtggactcttgc 7762 7781 SEQ ID NO: 2487 gcaagttaaagaaaatcag 14018 14037 1 3 SEQ ID NO: 2182 atgacagcattggtagagc 7839 7858 SEQ ID NO: 2488 gctcatctcctttcttcat 10200 10219 1 3 SEQ ID NO: 2183 tgaaagcattggtagagca 7840 7859 SEQ ID NO: 2489 tgctcatctcctttcttca 10199 10218 1 3 SEQ ID NO: 2184 gggttcactgttcctgaaa 7860 7879 SEQ ID NO: 2490 tttcaccatagaaggaccc 8951 8970 1 3 SEQ ID NO: 2185 tcaagaccatccttgggac 7879 7898 SEQ ID NO: 2491 gtccccctaacagatttga 7965 7984 1 3 SEQ ID NO: 2186 ccttgggaccatgcctgcc 7889 7908 SEQ ID NO: 2492 ggcaccagggctcggaagg 13970 13989 1 3 SEQ ID NO: 2187 ttcaggctcttcagaaagc 7921 7940 SEQ ID NO: 2493 gcttgaaggaattcttgaa 9580 9599 1 3 SEQ ID NO: 2188 ttcagataaacttcaaaga 7996 8015 SEQ ID NO: 2494 tcttcataagttcaatgaa 13175 13194 1 3 SEQ ID NO: 2189 acttcaaagacttaaaaaa 8005 8024 SEQ ID NO: 2495 ttttaacaaaagtggaagt 6821 6840 1 3 SEQ ID NO: 2190 atcccatccaggttttcca 8031 8050 SEQ ID NO: 2496 tggagaagcaaatctggat 9464 9483 1 3 SEQ ID NO: 2191 gaatttaccatccttaaca 8055 8074 SEQ ID NO: 2497 tgttgaagtgtctccattc 9881 9900 1 3 SEQ ID NO: 2192 cattccttcctttccaatt 8081 8100 SEQ ID NO: 2498 aattccacttttgagaatg 10406 10425 1 3 SEQ ID NO: 2193 ttgaccagatgctgaacag 8137 8156 SEQ ID NO: 2499 ctgttgacagatttatcaa 12924 12943 1 3 SEQ ID NO: 2194 aatcaccctgccagacttc 8225 8244 SEQ ID NO: 2500 gaagttctcaattttgatt 8514 8533 1 3 SEQ ID NO: 2195 tgaccttcacataccagaa 8312 8331 SEQ ID NO: 2501 ttcttctggaaaagggtca 8876 8895 1 3 SEQ ID NO: 2196 ttccagcttccccacatct 8331 8350 SEQ ID NO: 2502 agattctcagatgagggaa 6913 8932 1 3 SEQ ID NO: 2197 aagctatacagtattctga 8379 8398 SEQ ID NO: 2503 tcagatggcattgctgctt 11604 11623 1 3 SEQ ID NO: 2198 attctgaaaatccaatctc 8391 8410 SEQ ID NO: 2504 gagataaccgtgcctgaat 11544 11563 1 3 SEQ ID NO: 2199 tttcacattagatgcaaat 8414 8433 SEQ ID NO: 2505 attttgaaaaaaacagaaa 9730 9749 1 3 SEQ ID NO: 2200 caaatgctgacatagggaa 8428 8447 SEQ ID NO: 2506 ttccatcaccaatcctttg 9682 9881 1 3 SEQ ID NO: 2201 gagagtccaaattagaagt 8500 8519 SEQ ID NO: 2507 actttacttcccaactctc 13402 13421 1 3 SEQ ID NO: 2202 agagtccaaattagaagtt 8501 8520 SEQ ID NO: 2508 aactttacttcccaactct 13401 13420 1 3 SEQ ID NO: 2203 tctcaattttgattttcaa 8519 8538 SEQ ID NO: 2509 ttgattcccttttttgaga 11529 11548 1 3 SEQ ID NO: 2204 caattttgattttccagca 8522 8541 SEQ ID NO: 2510 tgctgaatccaaaagattg 13652 13871 1 3 SEQ ID NO: 2205 aatgcacaactctcaaacc 8541 8560 SEQ ID NO: 2511 ggtttatcaaggggccatt 12452 12471 1 3 SEQ ID NO: 2206 agttctccagcaagtccct 8596 8615 SEQ ID NO: 2512 aggttccatcgtgcaaact 11380 11399 1 3 SEQ ID NO: 2207 agtacctgagaacggagca 8608 8627 SEQ ID NO: 2513 tgctccaggagaacttact 13772 13791 1 3 SEQ ID NO: 2208 tcaaacacagtggcaagtt 8670 8689 SEQ ID NO: 2514 aactctcaagtcaagttga 13414 13433 1 3 SEQ ID NO: 2209 acaatcagcttaccctgga 8743 8762 SEQ ID NO: 2515 tccattctgaatatattgt 13372 13391 1 3 SEQ ID NO: 2210 ctggatagcaacactaaat 8757 8776 SEQ ID NO: 2516 attttctgaacttccccag 12694 12713 1 3 SEQ ID NO: 2211 ctgacctgcgcaacgagat 8821 8840 SEQ ID NO: 2517 atctgatgaggaaactcag 12251 12270 1 3 SEQ ID NO: 2212 agatgagggaacacatgaa 8921 8940 SEQ ID NO: 2518 ttcatgtccctagaaatct 10030 10049 1 3 SEQ ID NO: 2213 tcaacttttctaaacttga 9052 9071 SEQ ID NO: 2519 tcaaggataacgtgtttga 12610 12629 1 3 SEQ ID NO: 2214 ttctaaacttgaaattcaa 9059 9078 SEQ ID NO: 2520 ttgatgatgctgtcaagaa 7300 7319 1 3 SEQ ID NO: 2215 gaaattcaatcacaagtcg 9069 9088 SEQ ID NO: 2521 cgacgaagaaactaatttc 13558 13577 1 3 SEQ ID NO: 2216 cactgtttggagaagggaa 9133 9152 SEQ ID NO: 2522 ttccagaaagcagccagtg 12498 12517 1 3 SEQ ID NO: 2217 actgtttggagaagggaag 9134 9153 SEQ ID NO: 2523 cttccccaaagagaccagt 2890 2909 1 3 SEQ ID NO: 2218 aattctcttttcttttcag 9213 9232 SEQ ID NO: 2524 ctgattactatgaaaaatt 13630 13649 1 3 SEQ ID NO: 2219 ttcttttcagcccagccat 9222 9241 SEQ ID NO: 2525 atggaaaagggaaagagaa 13488 13505 1 3 SEQ ID NO: 2220 tttgaaagttcgttttcca 9275 9294 SEQ ID NO: 2526 tggaagtgtcagtggcaaa 10372 10391 1 3 SEQ ID NO: 2221 cagggaagatagacttcct 9304 9323 SEQ ID NO: 2527 aggacctttcaaattcctg 9840 9859 1 3 SEQ ID NO: 2222 ataagtacaaccaaaattt 9397 9416 SEQ ID NO: 2528 aaatcaggatctgagttat 14030 14049 1 3 SEQ ID NO: 2223 acaacgagaacattatgga 9427 9448 SEQ ID NO: 2529 tccattctgaatatattgt 13372 13391 1 3 SEQ ID NO: 2224 aggaataaatggagaagca 9455 9474 SEQ ID NO: 2530 tgctggaattgtcattcct 11726 11745 1 3 SEQ ID NO: 2225 agcaaatctggatttctta 9470 9489 SEQ ID NO: 2531 taagttctctgtacctgct 11711 11730 1 3 SEQ ID NO: 2226 tcctttaacaattcctgaa 9494 9513 SEQ ID NO: 2532 ttcacaacgagcttcagga 13198 13217 1 3 SEQ ID NO: 2227 tttaacaattcctgaaatg 9497 9516 SEQ ID NO: 2533 catttgatttaagtgtaaa 9613 9832 1 3 SEQ ID NO: 2228 acacaataatcacaactcc 9526 9545 SEQ ID NO: 2534 ggagacagcatcttcgtgt 11203 11222 1 3 SEQ ID NO: 2229 aagatttctctctatggga 9553 9572 SEQ ID NO: 2535 tcccagaaaacctcttctt 3928 3947 1 3 SEQ ID NO: 2230 gaaaaaacaggcttgaagg 9570 9589 SEQ ID NO: 2536 ccttttacaattcattttc 13013 13032 1 3 SEQ ID NO: 2231 ttgaaggaattcttgaaaa 9582 9601 SEQ ID NO: 2537 ttttgagaatgaatttcaa 10414 10433 1 3 SEQ ID NO: 2232 tgaaggaattcttgaaaac 9583 9602 SEQ ID NO: 2538 gttttggctgataaattca 11283 11302 1 3 SEQ ID NO: 2233 agctcagtataagaaaaac 9632 9651 SEQ ID NO: 2539 gtttgataagtacaaagct 9797 9816 1 3 SEQ ID NO: 2234 tcaaatcctttgacaggca 9712 9731 SEQ ID NO: 2540 tgcctgagcagaccattga 11680 11699 1 3 SEQ ID NO: 2235 atgaaacaaaaattaagtt 9781 9800 SEQ ID NO: 2541 aactttgcactatgttcat 12754 12773 1 3 SEQ ID NO: 2236 aattcctggatacactgtt 9851 9870 SEQ ID NO: 2542 aacacatgaatcacaaatt 8930 8949 1 3 SEQ ID NO: 2237 ttccagttgtcaatgttga 9868 9887 SEQ ID NO: 2543 tcaaaacgagcttcaggaa 13199 13218 1 3 SEQ ID NO: 2238 aagtgtctccattcaccat 9886 9905 SEQ ID NO: 2544 atgggaagtataagaactt 4834 4853 1 3 SEQ ID NO: 2239 gtcagcatgcctagtttct 9942 9961 SEQ ID NO: 2545 agaaaaggcacaccttgac 11072 11091 1 3 SEQ ID NO: 2240 ctgccatgggcaatattac 10105 10124 SEQ ID NO: 2546 gtaagaaaatacagagcag 6432 6451 1 3 SEQ ID NO: 2241 tgaataccaatgctgaact 10159 10178 SEQ ID NO: 2547 agttgaaggagactattca 7216 7235 1 3 SEQ ID NO: 2242 tattgttgctcatctcctt 10193 10212 SEQ ID NO: 2548 aaggaaacataaactaata 12881 12900 1 3 SEQ ID NO: 2243 tgttgctcatctcctttct 10196 10215 SEQ ID NO: 2549 atcaacacaatcttcaatg 12423 12442 1 3 SEQ ID NO: 2244 tctgtcattgatgcactgc 10224 10243 SEQ ID NO: 2550 cttggtacgagttactcaa 13920 13939 1 3 SEQ ID NO: 2245 ccacagctctgtctctgag 10297 10316 SEQ ID NO: 2551 agtgattacacttcctttc 8187 8206 1 3 SEQ ID NO: 2246 atttgtggagggtagtcat 10322 10341 SEQ ID NO: 2552 atgaagtagaccaacaaat 7153 7172 1 3 SEQ ID NO: 2247 atatggaagtgtcagtggc 10369 10388 SEQ ID NO: 2553 gccacactccaacgcatat 10770 10789 1 3 SEQ ID NO: 2248 tggaaataccaagtcaaaa 10445 10464 SEQ ID NO: 2554 ttttacaattcattttcca 13015 13034 1 3 SEQ ID NO: 2249 aagtcaaaacctactgtct 10455 10474 SEQ ID NO: 2555 agacctagtgattacactt 12851 12870 1 3 SEQ ID NO: 2250 actgtctcttcctccatgg 10467 10486 SEQ ID NO: 2556 ccatgcaagtcagcccagt 10916 10935 1 3 SEQ ID NO: 2251 cttcctccatggaatttaa 10474 10493 SEQ ID NO: 2557 ttaatcgagaggtatgaag 7140 7159 1 3 SEQ ID NO: 2252 attcttcaatgctgtactc 10504 10523 SEQ ID NO: 2558 gagttgagggtccgggaat 12234 12253 1 3 SEQ ID NO: 2253 ttgaccacaagcttagctt 10540 10559 SEQ ID NO: 2559 aagcgcacctcaatatcaa 12028 12047 1 3 SEQ ID NO: 2254 cctcacctcttacttttcc 10565 10584 SEQ ID NO: 2560 ggaactattgctagtgagg 10641 10660 1 3 SEQ ID NO: 2255 agctgcagggcacttccaa 10702 10721 SEQ ID NO: 2561 ttgggaagaagaggcagct 12281 12300 1 3 SEQ ID NO: 2256 ttccaaaattgatgatatc 10715 10734 SEQ ID NO: 2562 gatatacactagggaggaa 12737 12756 1 3 SEQ ID NO: 2257 gagaacatacaagcaaagc 10852 10871 SEQ ID NO: 2563 gcttggttttgccagtctc 2459 2478 1 3 SEQ ID NO: 2258 atggcaaatgtcagctctt 10889 10908 SEQ ID NO: 2564 aagaggtatttaaagccat 12952 12971 1 3 SEQ ID NO: 2259 tggcaaatgtcagctcttg 10890 10909 SEQ ID NO: 2565 caagaggtatttaaagcca 12951 12970 1 3 SEQ ID NO: 2260 ttgttcaggtccatgcaag 10906 10925 SEQ ID NO: 2566 cttgggggaggaggaacaa 14058 14077 1 3 SEQ ID NO: 2261 tgttcaggtccatgcaagt 10907 10926 SEQ ID NO: 2567 acttgggggaggaggaaca 14057 14076 1 3 SEQ ID NO: 2262 agttccttccatgatttcc 10932 10932 SEQ ID NO: 2568 ggaatctgatgaggaaact 12248 12267 1 3 SEQ ID NO: 2263 tgctaacactaagaaccag 10979 10998 SEQ ID NO: 2569 ctggatgtaaccaccagca 11178 11197 1 3 SEQ ID NO: 2264 actaagaaccagaagatca 10986 11005 SEQ ID NO: 2570 tgatcaagaacctgttagt 13339 13358 1 3 SEQ ID NO: 2265 ctaagaaccagaagatcag 10987 11006 SEQ ID NO: 2571 ctgatcaagaacctgttag 13338 13357 1 3 SEQ ID NO: 2266 cagaagatcagatggaaaa 10995 11014 SEQ ID NO: 2572 ttttcagaccaactctctg 13614 13633 1 3 SEQ ID NO: 2267 aaaaatgaagtccggattc 11010 11029 SEQ ID NO: 2573 gaatttgaaagttcgtttt 9272 9291 1 3 SEQ ID NO: 2268 gattcattctgggtctttc 11024 11043 SEQ ID NO: 2574 gaaaacctatgccttaatc 13158 13177 1 3 SEQ ID NO: 2269 aagaaaaggcacaccttga 11071 11090 SEQ ID NO: 2575 tcaaaacctactgtctctt 10458 10477 1 3 SEQ ID NO: 2270 aaggacacctaaggttcct 11107 11126 SEQ ID NO: 2576 aggacaccaaaataacctt 7564 7583 1 3 SEQ ID NO: 2271 ccagcattggtaggagaca 11191 11210 SEQ ID NO: 2577 tgtcaacaagtaccactgg 12362 12381 1 3 SEQ ID NO: 2272 ctttgtgtacaccaaaaac 11231 11250 SEQ ID NO: 2578 gtttttaaattgttgaaag 13140 13159 1 3 SEQ ID NO: 2273 ccatccctgtaaaagtttt 11269 11288 SEQ ID NO: 2579 aaaagggtcatggaaatgg 8885 8904 1 3 SEQ ID NO: 2274 tgatctaaattcagttctt 11324 11343 SEQ ID NO: 2580 aagatagtcagtctgatca 13326 13345 1 3 SEQ ID NO: 2275 aagaagctgagaacttcat 11424 11443 SEQ ID NO: 2581 atgagatcaacacaatctt 13102 13121 1 3 SEQ ID NO: 2276 tttgccctcaacctaccaa 11445 11464 SEQ ID NO: 2582 ttggtacgagttactcaaa 12633 12625 1 3 SEQ ID NO: 2277 cttgattcccttttttgag 11528 11547 SEQ ID NO: 2583 ctcaattttgattttcaag 8520 8539 1 3 SEQ ID NO: 2278 ttcacgcttccaaaaagtg 11583 11602 SEQ ID NO: 2584 cactcattgattttctgaa 12685 12704 1 3 SEQ ID NO: 2279 tgtttcagatggcattgct 11600 11519 SEQ ID NO: 2585 agcagattatgttgaaaca 11825 11844 1 3 SEQ ID NO: 2280 aatgcagtagccaacaaga 11631 11650 SEQ ID NO: 2586 tcttttcagcccagccatt 9223 9242 1 3 SEQ ID NO: 2281 ctgagcagaccattgagat 11683 11702 SEQ ID NO: 2587 atctgatgaggaaactcag 12251 12270 1 3 SEQ ID NO: 2282 tgagcagaccattgagatt 11684 11703 SEQ ID NO: 2588 aatctgatgaggaaactca 12250 12289 1 3 SEQ ID NO: 2283 ttgagattccctccattaa 11695 11714 SEQ ID NO: 2589 ttaatcttcataagttcaa 13171 13190 1 3 SEQ ID NO: 2284 acttggagtgccagtttga 11799 11818 SEQ ID NO: 2580 tcaattgggagagacaagt 6496 6515 1 3 SEQ ID NO: 2285 caaatttgaaggacttcag 11996 12015 SEQ ID NO: 2591 ctgagaacttcatcatttg 11430 11449 1 3 SEQ ID NO: 2286 agcccagcgttcaccgatc 12048 12067 SEQ ID NO: 2592 gatccaagtatagttggct 13278 13297 1 3 SEQ ID NO: 2287 cagcgttcaccgatctcca 12052 12071 SEQ ID NO: 2593 tggacctgcaccaaagctg 13952 13971 1 3 SEQ ID NO: 2288 ctccatctgcgctaccaga 12066 12085 SEQ ID NO: 2594 tctgatatacatcacggag 13703 13722 1 3 SEQ ID NO: 2289 atgaggaaactcagatcaa 12256 12275 SEQ ID NO: 2595 ttgagttgcccaccatcat 11659 11678 1 3 SEQ ID NO: 2290 aggcagcttctggcttgct 12292 12311 SEQ ID NO: 2596 agcaagtctttcctggcct 3010 3029 1 3 SEQ ID NO: 2291 tgaaagacaacgtgcccaa 12319 12338 SEQ ID NO: 2597 ttgggagagacaagtttca 6500 6519 1 3 SEQ ID NO: 2292 tatgattatgtcaacaagt 12354 12373 SEQ ID NO: 2598 actttgcactatgttcata 12755 12774 1 3 SEQ ID NO: 2293 cattaggcaaattgatgat 12467 12486 SEQ ID NO: 2599 atcaacacaatcttcaatg 13107 13126 1 3 SEQ ID NO: 2294 ttgactcaggaaggccaag 12576 12595 SEQ ID NO: 2600 cttggtacgagttactcaa 12632 12651 1 3 SEQ ID NO: 2295 gaaacctgggatatacact 12728 12747 SEQ ID NO: 2601 agtgattacacttcctttc 12857 12876 1 3 SEQ ID NO: 2296 tcctttcgagttaaggaaa 12869 12888 SEQ ID NO: 2602 tttctgccactgctcagga 13516 13535 1 3 SEQ ID NO: 2297 gccattcagtctctcaaga 12966 12985 SEQ ID NO: 2603 cctgcaccaaagctggcac 5794 5813 1 3 SEQ ID NO: 2298 gtgctacgtaatcttcagg 12993 13012 SEQ ID NO: 2604 cctgcaccaaagctggcac 13956 13975 1 3 SEQ ID NO: 2299 agctgaaagagatgaaatt 13057 13076 SEQ ID NO: 2605 aatttattcaaaacgagct 13192 13211 1 3 SEQ ID NO: 2300 aatttacttatcttattaa 13072 13091 SEQ ID NO: 2606 ttaacagaaatcttcaatt 13807 13826 1 3 SEQ ID NO: 2301 ttttaaattgttgaaagaa 13142 13161 SEQ ID NO: 2607 ttctctctatgggaaaaac 9558 9577 1 3 SEQ ID NO: 2302 taatcttcataagttcaat 13172 13191 SEQ ID NO: 2608 attgagattccctccatta 11694 11713 1 3 SEQ ID NO: 2303 atattttgatccaagtata 13271 13290 SEQ ID NO: 2609 tataagcagaagcacatat 13929 13948 1 3 SEQ ID NO: 2304 tgaaatattatgaacttga 13303 13322 SEQ ID NO: 2610 tcaaccttaatgattttca 8287 8306 1 3 SEQ ID NO: 2305 caatttctgcacagaaata 13434 13453 SEQ ID NO: 2611 tattcttcttttccaattg 13826 13845 1 3 SEQ ID NO: 2306 agaagattgcagagctttc 13501 13520 SEQ ID NO: 2612 gaaatcttcaatttcttct 13813 13832 1 3 SEQ ID NO: 2307 gaagaaaataatttctgat 13562 13581 SEQ ID NO: 2613 atcagttcagataaacttc 7991 8010 1 3 SEQ ID NO: 2308 ttgacctgtccattcaaaa 13872 13691 SEQ ID NO: 2614 ttttgagaatgaatttcaa 10414 10433 1 3 SEQ ID NO: 2309 tcaaaactaccacacattt 13686 13704 SEQ ID NO: 2615 aaattccttgacatgttga 7362 7381 1 3 SEQ ID NO: 2310 ttttttaaaagaaatcttc 13803 13822 SEQ ID NO: 2616 gaagtgtcagtggcaaaaa 10374 10393 1 3 SEQ ID NO: 2311 aggatctgagttattttgc 14035 14054 SEQ ID NO: 2617 gcaagggttcactgttcct 7856 7875 1 3 SEQ ID NO: 2312 tttgctaaacttgggggag 14049 14068 SEQ ID NO: 2618 ctccccaggacctttcaaa 9834 9853 1 3 # = Match Number B = Middle Matching Bases

TABLE 9 Selected palindromic sequences from human ApoB Start  End Start End Source Index Index Match Index Index # B SEQ ID NO: 2619 ggccattccagaagggaag 517 536 SEQ ID NO: 3948 cttccgttctgtaatggc 5803 6822 1 9 SEQ ID NO: 2620 tgccatctcgagagttcca 4107 4126 SEQ ID NO: 3949 tggaactctoccatggca 10884 10903 1 8 SEQ ID NO: 2621 catgtcaaacactttgtta 7064 7083 SEQ ID NO: 3950 taacaaattccttgacatg 7386 7385 1 8 SEQ ID NO: 2622 tttgttataaatcttattg 7076 7095 SEQ ID NO: 3951 caataagatcaatagcaaa 8898 9017 1 8 SEQ ID NO: 2623 tctggaaaagggtcatgga 8885 8907 SEQ ID NO: 3959 tccatgtcccattacaga 11384 11383 1 8 SEQ ID NO: 2624 cagctcttgttcaggtcca 10908 10927 SEQ ID NO: 3960 tggacctgcaccaaagctg 13960 13979 1 8 SEQ ID NO: 2625 ggaggttccccagctctgc 364 383 SEQ ID NO: 3961 gcagccctgggaaaactcc 6455 6474 1 7 SEQ ID NO: 2626 ctgttttgaagactctcca 1069 1108 SEQ ID NO: 3962 tggagggtagtcataacag 10335 10354 1 7 SEQ ID NO: 2627 agtggctgaaacgtgtgca 1305 1324 SEQ ID NO: 3963 tgcagagctttctgccact 13516 13535 1 7 SEQ ID NO: 2628 ccaaaatagaagggaatct 2076 2095 SEQ ID NO: 3964 aggattcctttgcctttgg 4008 4027 1 7 SEQ ID NO: 2629 tgaagagaagattgaattt 3628 3647 SEQ ID NO: 3965 aaattctcttttcttttca 9220 9239 1 7 SEQ ID NO: 2630 agtggtggcaacaccagca 4238 4257 SEQ ID NO: 3966 tgctagtgaggccaacact 10657 10676 1 7 SEQ ID NO: 2631 aaggctccacaagtcatca 5958 5977 SEQ ID NO: 3967 tgatgatatctggaacctt 10732 10751 1 7 SEQ ID NO: 2632 gtcagccaggtttatagca 7733 7752 SEQ ID NO: 3968 tgctaagaaccttactgac 7789 7808 1 7 SEQ ID NO: 2633 tgatatctggaaccttgaa 10735 10754 SEQ ID NO: 3969 ttcactgttcctgaaatca 7871 7890 1 7 SEQ ID NO: 2634 gtcaagttgagcaatttct 13431 13450 SEQ ID NO: 3970 agaaaaggcacaccttgac 11080 11099 1 7 SEQ ID NO: 2635 atccagatggaaaagggaa 13488 13507 SEQ ID NO: 3971 ttccaatttccctgtggat 3688 3707 1 7 SEQ ID NO: 2636 atttgtttgtcaaagaagt 4551 4570 SEQ ID NO: 3972 acttcagagaaatacaaat 11409 11428 4 6 SEQ ID NO: 2637 ctggaaaatgtcagcctgg 212 231 SEQ ID NO: 3973 ccagacttccgttteccag 8243 5282 2 6 SEQ ID NO: 2638 accaggaggttcttcttca 1737 1756 SEQ ID NO: 3974 tgaagtgtagtctcctggt 5097 5116 2 6 SEQ ID NO: 2639 aaagaagttctgaaagaat 1864 1983 SEQ ID NO: 3975 attccatcacaaatccttt 9669 9688 2 6 SEQ ID NO: 2640 gctacagcttatggctcca 3578 3597 SEQ ID NO: 3976 tggatctaaatgcagtagc 11631 11650 2 6 SEQ ID NO: 2641 atcaatattgatcaatttg 6422 6441 SEQ ID NO: 3977 caaagaagtcaagattgat 4561 4580 2 6 SEQ ID NO: 2642 gaattatcttttaaaacat 7334 7353 SEQ ID NO: 3978 atgtgttaacaaaatattc 11502 11521 2 6 SEQ ID NO: 2643 cgcggcccgcgctgctggc 138 157 SEQ ID NO: 3979 gccagaagtgagatcctcg 3515 3534 1 6 SEQ ID NO: 2644 acaactatgaggctgagag 279 298 SEQ ID NO: 3980 ctctgagcaacaaattagt 10317 10338 1 6 SEQ ID NO: 2645 gctgagagttccagtggag 290 309 SEQ ID NO: 3981 ctccatggcaaatgtcagc 10893 10912 1 6 SEQ ID NO: 2645 tgaagaaaaccaagaactc 456 475 SEQ ID NO: 3982 gagtcattgaggttcttca 4937 4956 1 6 SEQ ID NO: 2647 cctacttacatcctgaaca 566 585 SEQ ID NO: 3983 tgttcataagggaggtagg 12774 12793 1 6 SEQ ID NO: 2848 ctacttacatcctgaacat 567 586 SEQ ID NO: 3984 atgttcataagggaggtag 12773 12792 1 6 SEQ ID NO: 2649 gagacagaagaagccaagc 623 642 SEQ ID NO: 3985 gcttggttttgccagtctc 2467 2486 1 8 SEQ ID NO: 2650 cactcactttaccgtcaag 679 698 SEQ ID NO: 3986 cttgaacacaaagtcagtg 8008 6027 1 6 SEQ ID NO: 2651 ctgatcagcagcagccagt 830 849 SEQ ID NO: 3967 actgggaagtgcttatcag 5245 5264 1 6 SEQ ID NO: 2652 actggacgctaagaggaag 862 881 SEQ ID NO: 3988 cttccccaaagagaccagt 2898 2917 1 6 SEQ ID NO: 2653 agaggaagcatgtggcaga 873 892 SEQ ID NO: 3989 tctggcatttactttctct 5929 5948 1 6 SEQ ID NO: 2654 tgaagactctccaggaact 1096 1114 SEQ ID NO: 3990 agttgaaggagactattca 7224 7243 1 6 SEQ ID NO: 2655 ctctgagcaaaatatccag 1129 1148 SEQ ID NO: 3991 ctggttactgagctgagag 1169 1188 1 6 SEQ ID NO: 2656 atgaagcagtcacatctct 1197 1216 SEQ ID NO: 3992 agagctgccagtccttcat 10024 10043 1 6 SEQ ID NO: 2657 ttgccacagctgattgagg 1217 1236 SEQ ID NO: 3993 cctcctacagtggtggcaa 4230 4249 1 6 SEQ ID NO: 2658 agctgattgaggtgtocag 1224 1243 SEQ ID NO: 3994 ctggattccacatgcagct 11855 11874 1 6 SEQ ID NO: 2659 tgctccactcacatcctcc 1286 1305 SEQ ID NO: 3995 ggaggctttaagttcagca 7609 7628 1 6 SEQ ID NO: 2660 tgaaacgtgtgcatgccaa 1311 1330 SEQ ID NO: 3996 ttgggagagacaagtttca 6508 6527 1 6 SEQ ID NO: 2661 gacattgctaattacctga 1511 1530 SEQ ID NO: 3997 tcagaagctaagcaatgtc 7240 7259 1 6 SEQ ID NO: 2662 ttcttcttcagactttcct 1746 1765 SEQ ID NO: 3998 aggagagtccaaattagaa 8506 8525 1 6 SEQ ID NO: 2663 ccaatatcttgaactcaga 1911 1930 SEQ ID NO: 3999 tctgaattcattcaattgg 6493 6512 1 6 SEQ ID NO: 2664 aaagttagtgaaagaagtt 1954 1973 SEQ ID NO: 4000 aactaccctcactgccttt 2140 2159 1 8 SEQ ID NO: 2665 aagttagtgaaagaagttc 1955 1974 SEQ ID NO: 4001 gaacctctggcatttactt 5924 5943 1 6 SEQ ID NO: 2666 aaagaagttctgaaagaat 1954 1983 SEQ ID NO: 4002 attctctggtaactacttt 5490 5509 1 6 SEQ ID NO: 2667 tttggctataccaaagatg 2330 2349 SEQ ID NO: 4003 vatcttaggcactgacaaa 5005 5024 1 6 SEQ ID NO: 2668 tgttgagaagctgattaaa 2389 2408 SEQ ID NO: 4004 tttagccatcggctcaaca 5708 5727 1 6 SEQ ID NO: 2669 caggaagggctcaaagaat 2569 2588 SEQ ID NO: 4005 attcctttaacaattcctg 9500 9519 1 6 SEQ ID NO: 2670 aggaagggctcaaagaatg 2570 2569 SEQ ID NO: 4006 cattcctttaacaattcct 9499 9518 1 6 SEQ ID NO: 2671 gaagggctcaaagaatgac 2572 2591 SEQ ID NO: 4007 gtcagtcttcaggctcttc 7922 7941 1 6 SEQ ID NO: 2672 caaagaatgacttttttct 2580 2599 SEQ ID NO: 4008 agaaggatggcattttttg 14006 14027 1 6 SEQ ID NO: 2673 catggagaatgcctttgaa 2811 2830 SEQ ID NO: 4009 ttcagagccaaagtccatg 7127 7146 1 6 SEQ ID NO: 2674 ggagccaaggctggagtaa 2687 2706 SEQ ID NO: 4010 ttactccaacgccagctcc 3058 3077 1 6 SEQ ID NO: 2875 tcattccttccccaaagag 2892 2911 SEQ ID NO: 4011 ctctctggggcatctatga 5147 5166 1 6 SEQ ID NO: 2876 acctatgagctccagagag 3173 3192 SEQ ID NO: 4012 ctctcaagaccacagaggt 12984 13003 1 6 SEQ ID NO: 2677 gggcaaaacgtcttacaga 3373 3392 SEQ ID NO: 4013 tctgaaagacaacgtgccc 12325 12344 1 6 SEQ ID NO: 2678 accctggacattcagaaca 3395 3414 SEQ ID NO: 4014 tgttgctaaggttcagggt 5683 5702 1 6 SEQ ID NO: 2679 atgggcgacctaagttgtg 3437 3456 SEQ ID NO: 4015 cacaaattagtttcaccat 8949 8968 1 6 SEQ ID NO: 2680 gatgaagagaagattgaat 3626 3645 SEQ ID NO: 4016 attccagcttccccacatc 8338 8357 1 6 SEQ ID NO: 2881 caatgtagataccaaaaaa 3804 3683 SEQ ID NO: 4017 ttttttggaaatgccattg 8651 8670 1 6 SEQ ID NO: 2682 gtagataccaaaaaaatga 3668 3887 SEQ ID NO: 4018 tcatgtgatgggtctctac 4379 4398 1 6 SEQ ID NO: 2683 gcttcagttcatttggact 4517 4536 SEQ ID NO: 4019 agtcaagaaggacttaagc 5312 5331 1 6 SEQ ID NO: 2684 tttgtttgtcaaagaagtc 4552 4571 SEQ ID NO: 4020 gacttcagagaaatacaaa 11408 11427 1 6 SEQ ID NO: 2685 ttgtttgtcaaagaagtca 4563 4672 SEQ ID NO: 4021 tgacttcagagaaatacaa 11407 11426 1 6 SEQ ID NO: 2686 tggcaatgggaaactcgct 5854 5873 SEQ ID NO: 4022 agcgagaatcaccctgcca 8227 8246 1 6 SEQ ID NO: 2687 aacctctggcatttacttt 5925 5944 SEQ ID NO: 4023 aaaggagatgtcaagggtt 10607 10828 1 6 SEQ ID NO: 2888 catttactttctctcatga 5934 5953 SEQ ID NO: 4024 tcatttgaaagaataaatg 7034 7053 1 6 SEQ ID NO: 2889 aaagtcagtgccctgctta 6017 6036 SEQ ID NO: 4025 taagaaccttactgacttt 7792 7811 1 6 SEQ ID NO: 2690 tcccattttttgagacctt 6330 6349 SEQ ID NO: 4026 aaggacttcaggataggga 12012 12031 1 6 SEQ ID NO: 2691 catcaatattgatcaattt 6421 6440 SEQ ID NO: 4027 aaattaaaaagtgttgatg 6740 6759 1 6 SEQ ID NO: 2692 taaagatagttatgattta 6673 6692 SEQ ID NO: 4028 taaaccaaaacttggttta 9027 9046 1 6 SEQ ID NO: 2693 tattgatgaaatcattgaa 6721 6740 SEQ ID NO: 4029 ttcaaagacttaaaaaata 8015 8034 1 6 SEQ ID NO: 2694 atgatctacatttgtttat 6798 6817 SEQ ID NO: 4030 ataaagaaattaaagtcat 7386 7407 1 6 SEQ ID NO: 2695 agagacacatacagaatat 6927 6946 SEQ ID NO: 4031 atatattgtcagtgcctct 13390 13409 1 6 SEQ ID NO: 2696 gacacatacagaatataga 6930 6949 SEQ ID NO: 4032 tctaaattcagttcttgtc 11336 11354 1 6 SEQ ID NO: 2697 agcatgtcaaacactttgt 7062 7081 SEQ ID NO: 4033 acaaagtcagtgccctgct 6015 6034 1 6 SEQ ID NO: 2698 tttttagaggaaaccaagg 7523 7542 SEQ ID NO: 4034 cctttgtgtacaccaaaaa 11238 11257 1 6 SEQ ID NO: 2699 ttttagaggaaaccaaggc 7524 7543 SEQ ID NO: 4035 gcctttgtgtacaccaaaa 11237 11256 1 6 SEQ ID NO: 2700 ggaagatagacttcctgaa 9315 9334 SEQ ID NO: 4036 ttcagaaatactgttttcc 12832 12851 1 8 SEQ ID NO: 2701 cactgtttctgagtcccag 9342 9361 SEQ ID NO: 4037 ctgggacctaccaagagtg 12531 12550 1 6 SEQ ID NO: 2702 cacaaatcctttggctgtg 9676 9695 SEQ ID NO: 4038 cacatttcaaggaattgtg 10071 10090 1 6 SEQ ID NO: 2703 ttcctggatacactgttcc 9861 9880 SEQ ID NO: 4039 ggaactgttgactcaggaa 12577 12596 1 6 SEQ ID NO: 2704 gaaatctcaagctttctct 10050 1006 SEQ ID NO: 4040 agagccaggtcgagctttc 11052 11071 1 6 SEQ ID NO: 2705 tttcttcatcttcatctgt 10218 10237 SEQ ID NO: 4041 acagctgaaagagatgaaa 13063 13082 1 6 SEQ ID NO: 2706 tctaccgctaaaggagcag 10529 10548 SEQ ID NO: 4042 ctgcacgctttgaggtaga 11769 11788 1 6 SEQ ID NO: 2707 ctaccgctaaaggagcagt 10530 10549 SEQ ID NO: 4043 actgcacgctttgaggtag 11768 11787 1 6 SEQ ID NO: 2708 agggcctctttttcaccaa 10839 10858 SEQ ID NO: 4044 ttggccaggaagtggccct 10985 10984 1 6 SEQ ID NO: 2709 ttctccatccctgtaaaag 11273 11292 SEQ ID NO: 4045 ctttttcaccaacggagaa 10846 10665 1 6 SEQ ID NO: 2710 gaaaaacaaagcagattat 11824 11843 SEQ ID NO: 4048 ataaactgcaagatttttc 13608 13627 1 6 SEQ ID NO: 2711 actcactcattgattttct 12890 12709 SEQ ID NO: 4047 agaaaatcaggatctgagt 14035 14054 1 6 SEQ ID NO: 2712 taaactaatagatgtaatc 12896 12917 SEQ ID NO: 4045 gattaccaccagcsgttta 13586 13605 1 6 SEQ ID NO: 2713 caaaacgagcttcaggaag 13208 13227 SEQ ID NO: 4049 cttcgtgaagaatattttg 13268 13287 1 6 SEQ ID NO: 2714 tggaataatgctcagtgtt 2374 2393 SEQ ID NO: 4050 aacacttacttgaattcca 10670 10689 3 5 SEQ ID NO: 2715 gatttgaaatccaaagaag 2406 2427 SEQ ID NO: 4051 cttcagagaaatacaaatc 11410 11429 3 5 SEQ ID NO: 2716 atttgaaatccaaagaagt 2409 2428 SEQ ID NO: 4052 acttcagagaaatacaaat 11409 11428 3 5 SEQ ID NO: 2717 atcaacagccgcttctttg 998 1017 SEQ ID NO: 4053 caaagaagtcaagattgat 4561 4580 2 5 SEQ ID NO: 2718 tgttttgaagactctccag 1090 1109 SEQ ID NO: 4054 ctggaaagttaaaacaaca 6963 6982 2 5 SEQ ID NO: 2719 cccttctgatagatgtggt 1332 1351 SEQ ID NO: 4055 accaaagctggcaccaggg 13969 13988 2 5 SEQ ID NO: 2720 tgagcaagtgaagaacttt 1876 1895 SEQ ID NO: 4058 aaagccattcagtctctca 12971 12990 2 5 SEQ ID NO: 2721 atttgaaatccaaagaagt 2408 2428 SEQ ID NO: 4057 acttttctaaacttgaaat 9063 9082 2 5 SEQ ID NO: 2722 atccaaaagaagtcccggaa 2416 2435 SEQ ID NO: 4058 ttccggggaaacctgtgat 12729 12746 2 5 SEQ ID NO: 2723 agagcctacctccgcatct 2438 2457 SEQ ID NO: 4059 agatggtacgttagcctct 11929 11948 2 5 SEQ ID NO: 2724 aatgcctttgaactcccca 2818 2637 SEQ ID NO: 4060 tgggaactacaatttcatt 7020 7039 2 5 SEQ ID NO: 2725 gaagtccaaattccggatt 3305 3324 SEQ ID NO: 4081 aatcttcaatttattcttc 13823 13842 2 5 SEQ ID NO: 2726 tgcaagcagaagccagaag 3504 3523 SEQ ID NO: 4062 cttcaggttccatcgtgca 11384 11403 2 5 SEQ ID NO: 2727 gaagagaagattgaatttg 3629 3848 SEQ ID NO: 4003 caaaacctactgtctcttc 10467 10486 2 5 SEQ ID NO: 2728 atgctaaaggcacatatgg 4605 4624 SEQ ID NO: 4054 ccatatgaaagtcaagcat 12664 12683 2 5 SEQ ID NO: 2729 tccctcacctccacctctg 4745 4784 SEQ ID NO: 4005 cagattctcagagaggga 8920 8939 2 5 SEQ ID NO: 2730 atttacagctctgacaat 5435 5454 SEQ ID NO: 4068 acttttctaacttgaaat 9063 9062 2 5 SEQ ID NO: 2731 aggagcctaccaaaataat 5802 5821 SEQ ID NO: 4087 attatgttgaaacagtcct 11838 11857 2 5 SEQ ID NO: 2732 aaagctgaagcacatcaat 6409 6428 SEQ ID NO: 4068 attgttgctcatctccttt 10202 10221 2 5 SEQ ID NO: 2733 ctgctggaaacaacgagaa 9426 9445 SEQ ID NO: 4069 ttctgattaccaccagcag 13582 13601 2 5 SEQ ID NO: 2734 ttgaaggaattcttgaaaa 9580 8808 SEQ ID NO: 4070 ttttaaaaaaaatcttcaa 13813 13832 2 5 SEQ ID NO: 2735 gaagtaaaagaaaattttg 10751 10770 SEQ ID NO: 4071 caaaacctactgtctcttc 10467 10486 2 5 SEQ ID NO: 2736 tgaagaagatggcaaattt 11882 12011 SEQ ID NO: 4072 aaatgtcagctcttgttca 10902 10921 2 5 SEQ ID NO: 2737 aggatctgagttattttgc 14043 14082 SEQ ID NO: 4073 gcaagtcagcccagttcct 10928 10947 2 5 SEQ ID NO: 2738 gtgcccttctcggtttgctg 26 45 SEQ ID NO: 4074 cagccattgacatgagcac 5748 5767 1 5 SEQ ID NO: 2739 ggcgctgcctgcgctgctg 154 173 SEQ ID NO: 4075 cagctccacagactccgcc 3070 3089 1 5 SEQ ID NO: 2740 ctgcgctgctgctgctgct 162 181 SEQ ID NO: 4076 agcagaaggtgcgaagcag 3232 3251 1 5 SEQ ID NO: 2741 gctgctggcgggcgccagg 178 197 SEQ ID NO: 4077 cctggattccacatgcagc 11854 11873 1 5 SEQ ID NO: 2742 aagaggaaatgctggaaaa 201 220 SEQ ID NO: 4078 tttttcttcactacatctt 2592 2611 1 5 SEQ ID NO: 2743 ctggaaaatgtcagcctgg 212 231 SEQ ID NO: 4079 ccagacttccacatcocag 3923 3042 1 5 SEQ ID NO: 2744 tggagtccctgggactgct 304 323 SEQ ID NO: 4080 agcatgcctagtttctcca 9953 9972 1 5 SEQ ID NO: 2745 ggagtccctgggactgctg 305 324 SEQ ID NO: 4081 cagcatgcctagtttctcc 9952 9971 1 5 SEQ ID NO: 2746 tgggactgctgattcaaga 313 332 SEQ ID NO: 4082 tcttccatcacttgaccca 2050 2069 1 5 SEQ ID NO: 2747 ctgctcattcaagaagtgc 318 337 SEQ ID NO: 4083 gcacaccttgacattgcag 11087 11106 1 5 SEQ ID NO: 2748 tgccaccaggatcaactgc 334 353 SEQ ID NO: 4084 gcaggctgaactggtggca 2725 2744 1 5 SEQ ID NO: 2749 gccaccaggatcaactgca 335 354 SEQ ID NO: 4085 tgcaggetgaactagtagc 2724 2743 1 5 SEQ ID NO: 2750 tgcaaggttgagctggagg 360 389 SEQ ID NO: 4086 cctccacctctgatctgca 4752 4771 1 5 SEQ ID NO: 2751 caaggttgagctggaggtt 352 371 SEQ ID NO: 4089 aacccctacatgaacttg 13763 13782 1 5 SEQ ID NO: 2752 ctctgcagcttcatcctga 377 386 SEQ ID NO: 4090 tcaggaagcttctcaagag 13219 13238 1 5 SEQ ID NO: 2753 cagcttcatcctgaagacc 382 401 SEQ ID NO: 4091 ggtcttgagttaaatgctg 4985 5004 1 5 SEQ ID NO: 2754 gcttcatcctgaagaccag 384 403 SEQ ID NO: 4092 ctggacgctaagaggaapc 863 882 1 5 SEQ ID NO: 2755 tcatcctgaagaccagcca 387 406 SEQ ID NO: 4093 tggcatggcattatgatga 3612 3631 1 5 SEQ ID NO: 2755 gaaaaccaagaactctgag 460 479 SEQ ID NO: 4094 ctcaaccttaatgatttttc 8294 8313 1 5 SEQ ID NO: 2767 agaactctgaggagtttgc 468 487 SEQ ID NO: 4095 gcaaggctatacagtattct 8385 5404 1 5 SEQ ID NO: 2758 tctgaggagtttgctgcag 473 492 SEQ ID NO: 4090 ctgcaggggatcccccaga 2534 2553 1 5 SEQ ID NO: 2759 tttgctgcagccatgtcca 482 501 SEQ ID NO: 4097 tggaagtgtcagtggcaaa 10380 10399 1 5 SEQ ID NO: 2760 caagaggggcatcatttct 586 805 SEQ ID NO: 4098 agaataaatgacgttcttg 7043 7062 1 5 SEQ ID NO: 2761 tcactttaccgtcaacacg 682 701 SEQ ID NO: 4099 cgtctacactatcatgtga 4368 4387 1 5 SEQ ID NO: 2762 tttaccgtcaacacgagga 688 705 SEQ ID NO: 4100 tccttgacatgttgataaa 7374 7393 1 5 SEQ ID NO: 2763 cactggacgctaagaggaa 861 880 SEQ ID NO: 4101 ttccagaaagcagccagtg 12506 12525 1 5 SEQ ID NO: 2764 aggaagcatgtggcagaag 875 894 SEQ ID NO: 4102 cttcatacacattaatcct 9995 10015 1 5 SEQ ID NO: 2765 caaggagcaacacctcttc 901 920 SEQ ID NO: 4103 gaagtagtactgcatcttg 6843 6862 1 5 SEQ ID NO: 2766 acagactttgaaacttgaa 967 986 SEQ ID NO: 4104 ttcaattcttcaatgctgt 10508 10527 1 5 SEQ ID NO: 2767 tgatgaagcagtcacatct 1195 1214 SEQ ID NO: 4105 agatttgaggattccatca 7984 8003 1 5 SEQ ID NO: 2768 agcagtcacatctctcttg 1201 1220 SEQ ID NO: 4106 caaggagaaactgactgct 6532 8551 1 5 SEQ ID NO: 2769 ccagccccatcactttaca 1239 1258 SEQ ID NO: 4107 tgtagtctcctggtgctgg 5102 5121 1 5 SEQ ID NO: 2770 ctccactcacatcctccag 1288 1307 SEQ ID NO: 4108 ctggagcttagtaatggag 8717 8736 1 5 SEQ ID NO: 2771 catgccaacccccttctga 1322 1341 SEQ ID NO: 4109 tcagatgagggaacacatg 8927 8946 1 5 SEQ ID NO: 2772 gagagatcttcaacatggc 1398 1417 SEQ ID NO: 4110 gccaccctggaactctctc 10877 10896 1 5 SEQ ID NO: 2773 tcaacatggcgagggatca 1407 1428 SEQ ID NO: 4111 tgatcccacctctcattga 2973 2992 1 5 SEQ ID NO: 2774 ccaccttgtatgcgctgag 1437 1456 SEQ ID NO: 4112 ctcagggatctgaaggtgg 8195 8214 1 5 SEQ ID NO: 2775 gtcaacaactatcataaga 1463 1482 SEQ ID NO: 4113 tcttgagttaaatgctgac 4987 5006 1 5 SEQ ID NO: 2776 tggacattgctaattacct 1509 1528 SEQ ID NO: 4114 aggtatattcgaaagtcca 12807 12826 1 5 SEQ ID NO: 2777 ggacattgctaattacctg 1510 1529 SEQ ID NO: 4115 caggtatattcgaaagtcc 12806 12825 1 5 SEQ ID NO: 2778 ttctgcgggtcattggaaa 1581 1600 SEQ ID NO: 4116 tttcacatgccaaggagaa 6522 6541 1 5 SEQ ID NO: 2779 ccagaactcaagtcttcaa 1628 1647 SEQ ID NO: 4117 ttgaagtgtagtctcctgg 5096 5115 1 5 SEQ ID NO: 2780 agtcttcaatcctgaaatg 1638 1657 SEQ ID NO: 4118 catttctgattggtggact 7765 7784 1 5 SEQ ID NO: 2781 tgagcaagtgaagaacttt 1876 1895 SEQ ID NO: 4119 aaagtgccacttttactca 6191 6210 1 5 SEQ ID NO: 2782 agcaagtgaagaactttgt 1878 1897 SEQ ID NO: 4120 acaaagtcagtgccctgct 6015 6034 1 5 SEQ ID NO: 2783 tctgaaagaatctcaactt 1872 1991 SEQ ID NO: 4121 aagaccataatggttcaga 12819 12838 1 5 SEQ ID NO: 2784 actgtcatggagttcagaa 1994 2013 SEQ ID NO: 4122 ttctgaatatattgtcagt 13384 13403 1 5 SEQ ID NO: 2785 acttgacccagcctcagcc 2069 2078 SEQ ID NO: 4123 ggctcaccctgagagaagt 12399 12418 1 5 SEQ ID NO: 2786 tccaaataactaccttcct 2104 2123 SEQ ID NO: 4124 aggaagatatgaagatgga 4720 4739 1 5 SEQ ID NO: 2787 actaccctcactgaatttg 2141 2180 SEQ ID NO: 4126 caaatttgtggaggtagt 10327 10348 1 5 SEQ ID NO: 2788 ttggatttgcttcagctga 2157 2176 SEQ ID NO: 4126 tcagtataagtacaaccaa 9400 9419 1 5 SEQ ID NO: 2789 ttggaagctctttttggga 2219 2239 SEQ ID NO: 4127 tcccgattcacgcttccaa 11585 11604 1 5 SEQ ID NO: 2790 ggaagctctttttgggaag 2221 2240 SEQ ID NO: 4128 cttcagaaagctaccttcc 7937 7956 1 5 SEQ ID NO: 2791 tttttcccagacagtgtca 2246 2285 SEQ ID NO: 4129 tgaccttctctaacaaaa 4884 4903 1 5 SEQ ID NO: 2792 agacagtgtcaacaaagct 2254 2273 SEQ ID NO: 4130 agcttggttttgccagtct 2465 2485 1 5 SEQ ID NO: 2793 ctttggctataccaaagat 2329 2349 SEQ ID NO: 4131 atctogtgtctaggaaaag 5976 5995 1 5 SEQ ID NO: 2794 caaagatgataaacatgag 2341 2390 SEQ ID NO: 4132 ctcaaggataacgtgtttg 12617 12636 1 5 SEQ ID NO: 2795 gatatggtaaatggaaiaa 2383 2382 SEQ ID NO: 4133 ttatcttattaattatatc 13067 13106 1 5 SEQ ID NO: 2796 ggaataatgctcagtgttg 2375 2394 SEQ ID NO: 4134 caacacttacttgaattcc 10669 10688 1 5 SEQ ID NO: 2797 tttgaaatccaaagaagtc 2410 2429 SEQ ID NO: 4135 gacttcagagaaatacaaa 11408 11427 1 5 SEQ ID NO: 2798 gatcccccagatgattgga 2542 2561 SEQ ID NO: 4138 tccaatttccctgtggatc 3689 3708 1 5 SEQ ID NO: 2798 cagatgattggagaggtca 2549 2568 SEQ ID NO: 4137 tgaccacacaaacagtctg 5371 5390 1 5 SEQ ID NO: 2800 agaatgacttttttcttca 2563 2802 SEQ ID NO: 4138 tgaagtccggattcattct 11023 11042 1 5 SEQ ID NO: 2801 gaactccccactggagctg 2627 2649 SEQ ID NO: 4139 cagctcaaccgtacagttc 11889 11808 1 5 SEQ ID NO: 2802 atatcttcatctggagtca 2860 2879 SEQ ID NO: 4140 tgacttcagtgcagaatat 11974 11993 1 5 SEQ ID NO: 2803 gtcattgctcccggagcca 2675 2694 SEQ ID NO: 4141 tggccccgtttaccatgac 5817 5836 1 5 SEQ ID NO: 2804 gctgaagtttatcattcct 2881 2900 SEQ ID NO: 4142 aggaggctttaagttcagc 7608 7627 1 5 SEQ ID NO: 2805 attccttccccaaagagac 2894 2913 SEQ ID NO: 4143 gtctcttcctccatggaat 10478 10497 1 5 SEQ ID NO: 2806 ctcattgagaacaggcagt 2994 3003 SEQ ID NO: 4144 actgactgcacgctttgag 11764 11763 1 5 SEQ ID NO: 2807 ttgagcagtattctgtcag 3160 3189 SEQ ID NO: 4145 ctgagagaagtgtcttcaa 12407 12428 1 5 SEQ ID NO: 2808 accttgtccagtgaagtcc 3293 3312 SEQ ID NO: 4146 ggacggtactgtcccaggt 12792 12811 1 5 SEQ ID NO: 2809 ccagtgaagtccaaattcc 3300 3319 SEQ ID NO: 4147 ggaaggcagagtttactgg 9156 9175 1 5 SEQ ID NO: 2810 acattcagaacaagaaaat 3402 3421 SEQ ID NO: 4148 atttcctaaagctggatgt 11175 11194 1 5 SEQ ID NO: 2811 gaaaaatcaagggtgttat 3471 3490 SEQ ID NO: 4149 ataaactgcaagatttttc 13608 13627 1 5 SEQ ID NO: 2812 aaatcaagggtgttatttc 3474 3493 SEQ ID NO: 4150 gaaacaatgcattagattt 9753 9772 1 5 SEQ ID NO: 2813 tggcattatgatgaagaga 3617 3838 SEQ ID NO: 4151 tctcccgtgtataatgcca 11789 11808 1 5 SEQ ID NO: 2814 aagagaagattgaatttga 3630 3649 SEQ ID NO: 4152 tcaaaacctactgtctctt 10466 10485 1 5 SEQ ID NO: 2515 aaatgacttccaatttccc 3681 3700 SEQ ID NO: 4153 gggaactacaatttcattt 7021 7040 1 5 SEQ ID NO: 2818 atgacttccaatttccctg 3883 3702 SEQ ID NO: 4154 caggctgattacgagtcat 4925 4944 1 5 SEQ ID NO: 2817 acttccaatttccctgtgg 3686 3706 SEQ ID NO: 4155 ccacgaaaaatatggaagt 10368 10387 1 5 SEQ ID NO: 2818 agttgcaatgagctcatgg 3811 3830 SEQ ID NO: 4156 ccatcagttcagataaact 7997 8016 1 5 SEQ ID NO: 2819 tttgcaagaccacctcaat 3868 3887 SEQ ID NO: 4157 attgacctgtccattcaaa 13879 13698 1 5 SEQ ID NO: 2820 gaaggagttcaacctccag 3892 3911 SEQ ID NO: 4158 ctggaattgtcattvvttc 117386 11756 1 5 SEQ ID NO: 2821 acttccacatcccagaaaa 3927 3046 SEQ ID NO: 4159 ttttaacaaaagtggaagt 6629 6648 1 5 SEQ ID NO: 2822 ctcttcttaaaaagcgatg 3947 3966 SEQ ID NO: 4160 catcactgocaaaggagag 8494 8513 1 5 SEQ ID NO: 2823 aaaagcgatggccgggtca 3956 3976 SEQ ID NO: 4161 tgactcactcattgatttt 12688 12707 1 5 SEQ ID NO: 2824 ttcctttgccttttggtgg 4011 4030 SEQ ID NO: 4162 ccacaaacaatgaagggaa 9264 9283 1 5 SEQ ID NO: 2825 caagtctgtgggattccat 4087 4106 SEQ ID NO: 4163 atgggaaaaaacaggcttg 9574 9593 1 5 SEQ ID NO: 2826 aagtccctacttttaccat 4125 4144 SEQ ID NO: 4164 atg9gsa9tat8808actt 4842 4881 1 5 SEQ ID NO: 2827 tgcctctcctgggtgttct 4167 4186 SEQ ID NO: 4165 agaaaaacaaacacaggca 9851 9870 1 5 SEQ ID NO: 2828 accagcacagaccatttca 4250 4269 SEQ ID NO: 4166 tgaagtgtagtctcctggt 5097 5116 1 5 SEQ ID NO: 2829 ccagcacagaccatttcag 4251 4270 SEQ ID NO: 4167 ctgaaatavaatcctctgg 5519 5536 1 5 SEQ ID NO: 2830 actatcatgtgatgggtct 4378 4394 SEQ ID NO: 4168 agacacctgattttatagt 7956 7975 1 5 SEQ ID NO: 2831 accacagatgtctgcttca 4504 4523 SEQ ID NO: 4169 tgaaggctgactctgtggt 4290 4309 1 5 SEQ ID NO: 2832 ccacagatgtctgcttcag 4505 4524 SEQ ID NO: 4170 ctgagcaacaaatttgtgg 10319 10338 1 5 SEQ ID NO: 2833 tttggactccaaaaagaaa 4528 4547 SEQ ID NO: 4171 tttctctcatgattacaaa 5941 5960 1 5 SEQ ID NO: 2834 tcaaagaagtcaagattga 4560 4879 SEQ ID NO: 4172 tcaaggataacgtgtttga 12616 12637 1 5 SEQ ID NO: 2835 atgagaactaogagctgac 4806 4825 SEQ ID NO: 4173 gtcagatattgttgctcat 10195 10214 1 5 SEQ ID NO: 2836 ttaaaatctgacaccaatg 4828 4848 SEQ ID NO: 4174 cattcattgaagatgttaa 7350 7369 1 6 SEQ ID NO: 2837 gaagtataagaactttgcc 4848 4863 SEQ ID NO: 4175 ggcaaatttgaaggacttc 12002 12021 1 5 SEQ ID NO: 2838 aagtataagaactttgcca 4847 4866 SEQ ID NO: 4176 tggcaaatttgaaggactt 12001 12020 1 5 SEQ ID NO: 2839 ttcttcagcctgctttctg 4949 4968 SEQ ID NO: 4177 cagaatccagatacaagaa 6892 5911 1 5 SEQ ID NO: 2840 ctggatcactaaattccca 4965 4984 SEQ ID NO: 4178 tgggtctttccagagccag 11041 11060 1 5 SEQ ID NO: 2841 aaattaatagtggtgctca 5022 5041 SEQ ID NO: 4179 tgagaaagcccaacaattt 6256 6275 1 5 SEQ ID NO: 2842 agtgcaacgaccaacttga 5081 5100 SEQ ID NO: 4180 tcaaattcctggatacact 9856 9875 1 5 SEQ ID NO: 2843 ctgggaagtgcttatcagg 5246 5265 SEQ ID NO: 4181 cctgaccttcacataccag 8318 8337 1 5 SEQ ID NO: 2844 gcaaaaacattttcaactt 5288 5305 SEQ ID NO: 4182 aagtaaaagaaaattttgc 10752 10771 1 5 SEQ ID NO: 2845 aaaaacattttcaacttca 5288 5307 SEQ ID NO: 4183 tgaagtaaaagaaaatttt 10750 10769 1 5 SEQ ID NO: 2846 tcagtcaagaaggacttaa 5310 5329 SEQ ID NO: 4184 ttaaggacttccattctga 13371 13390 1 5 SEQ ID NO: 2847 tcaaatgacatgatgggct 5333 5352 SEQ ID NO: 4185 agcccatcaatatcattga 6213 6232 1 5 SEQ ID NO: 2848 cacacaaacagtctgaaca 5375 5394 SEQ ID NO: 4186 tgtttcaactgoctttgtg 11227 11246 1 5 SEQ ID NO: 2849 tcttcaoaacttgacaaca 5417 5438 SEQ ID NO: 4187 tgttttcctatttocaaga 12843 12862 1 5 SEQ ID NO: 2850 caagttttataagcaaact 4449 5468 SEQ ID NO: 4188 agttattttgctaaacttg 14051 14070 1 5 SEQ ID NO: 2851 tggtaactactttaaacag 5496 5515 SEQ ID NO: 4189 ctgtttttagaggaaacca 7520 7539 1 5 SEQ ID NO: 2852 aacagtgacctgaaataca 5510 5529 SEQ ID NO: 4190 tgtatagcaaattaatgtt 5898 5917 1 5 SEQ ID NO: 2853 gggaaactacggctagaac 5552 5571 SEQ ID NO: 4191 gttccttccatgatttocc 10941 10960 1 5 SEQ ID NO: 2854 aacacatctatgccatctc 5628 5647 SEQ ID NO: 4192 gagacagcatcttcgtgtt 11212 11231 1 5 SEQ ID NO: 2855 tcagcaagctataaagcag 5660 5679 SEQ ID NO: 4193 ctgctaagaaccttactga 7788 7807 1 5 SEQ ID NO: 2856 gcagacactgttgctaagg 5675 5694 SEQ ID NO: 4194 cctttcaagcactgactgc 11754 11773 1 5 SEQ ID NO: 2857 tctggggagaacatactgg 5874 5693 SEQ ID NO: 4195 ccaggttttccacaccaga 8046 8065 1 5 SEQ ID NO: 2858 ttctctcatgattacaaag 5942 5961 SEQ ID NO: 4196 ctttttcaccaacggagaa 10846 10885 1 5 SEQ ID NO: 2859 ctgagcagacaggcacctg 6042 6061 SEQ ID NO: 4197 caggaggctttaagttcag 7607 7626 1 5 SEQ ID NO: 2860 caatttaacaagaatgaat 6074 6093 SEQ ID NO: 4198 attccttcctttacaattg 8090 8109 1 5 SEQ ID NO: 2861 tggacgaactctggctgac 6148 6167 SEQ ID NO: 4199 gtcagcccagttccttoca 10932 10951 1 5 SEQ ID NO: 2862 cttttactcagtgagccca 6200 6219 SEQ ID NO: 4200 tgggctaaacgtatgacag 7835 7854 1 5 SEQ ID NO: 2863 tcattgatgctttagagat 6225 6244 SEQ ID NO: 4201 atcttcataagttcaatga 13182 13201 1 5 SEQ ID NO: 2864 aaaaccaagatgttcactc 6303 6322 SEQ ID NO: 4202 gagtgaaaatgctgttttt 8638 8657 1 5 SEQ ID NO: 2865 aggaatcgacaaaccatta 6365 6384 SEQ ID NO: 4203 taatgattttcaagttcct 8302 8321 1 5 SEQ ID NO: 2866 tagttgtactggaaaacgt 6384 6403 SEQ ID NO: 4204 acgttagcctctaagacta 11938 11955 1 5 SEQ ID NO: 2867 ggaaaacgtacagagaaag 6394 6413 SEQ ID NO: 4205 cttttacaattcattttcc 13022 13041 1 5 SEQ ID NO: 2868 gaaaacgtacagagaaagc 6395 6414 SEQ ID NO: 4206 gctttctcttccacatttc 10060 10079 1 5 SEQ ID NO: 2869 aaagctgaagcacatcaat 6409 6428 SEQ ID NO: 4207 attgatgttagagtgcttt 6992 7011 1 5 SEQ ID NO: 2870 aagctgaagcacatcaata 6410 6429 SEQ ID NO: 4208 tattgatgttagagtgctt 6991 7010 1 5 SEQ ID NO: 2871 tgaagcacatcaatattga 6414 6433 SEQ ID NO: 4209 tcaaccttaatgattttca 6295 8314 1 5 SEQ ID NO: 2872 atcaatattgatcaatttg 6422 6441 SEQ ID NO: 4210 caaagccatcactgatgat 1668 1667 1 5 SEQ ID NO: 2873 taatgattatctgaattca 6484 6503 SEQ ID NO: 4211 tgaaatcattgaaaaatta 6727 6746 1 5 SEQ ID NO: 2874 gattatctgaattcattca 6488 6507 SEQ ID NO: 4212 tgaagtagctgagaaaatc 7102 7121 1 5 SEQ ID NO: 2875 aattgggagagacaagttt 6508 6525 SEQ ID NO: 4213 aaacattcctttaacaatt 9496 9515 1 5 SEQ ID NO: 2876 aaaatagctattgctaata 6701 6720 SEQ ID NO: 4214 tattgaaaatattgatttt 6814 6833 1 5 SEQ ID NO: 2877 aaaattaaaaagtcttgat 6739 6758 SEQ ID NO: 4215 atcatatccgtgaattttt 6765 6784 1 5 SEQ ID NO: 2878 ttgaaaatattgattttaa 6816 6835 SEQ ID NO: 4216 ttaatcttcataagttcaa 13179 13198 1 5 SEQ ID NO: 2879 agacatccagcacctagct 6846 6965 SEQ ID NO: 4217 agcttggttttgccagtct 2466 2465 1 5 SEQ ID NO: 2880 caatttcatttgaaagaat 7029 7048 SEQ ID NO: 4218 attccttcctttacaattg 8090 8109 1 5 SEQ ID NO: 2881 aggttttaatggataaatt 7182 7201 SEQ ID NO: 4219 aattgttgaaagaaaacct 13155 13174 1 5 SEQ ID NO: 2882 cagaagctaagcaatgtcc 7241 7260 SEQ ID NO: 4220 ggacaaggcccagaatctg 12553 12572 1 5 SEQ ID NO: 2883 taagataaaagattacttt 7270 7289 SEQ ID NO: 4221 aaagaaaacctatgcctta 13163 13182 1 5 SEQ ID NO: 2884 aaagattcatttgagaaat 7277 7296 SEQ ID NO: 4222 atttcttaaacattccttt 9489 9508 1 5 SEQ ID NO: 2885 gagaaattagttggattta 7289 7308 SEQ ID NO: 4223 taaagccattcagtctctc 12970 12989 1 5 SEQ ID NO: 2886 atttattgatgatgctgtc 7303 7322 SEQ ID NO: 4224 gacatgttgataaagaaat 7379 7398 1 5 SEQ ID NO: 2887 gaattatcttttaaaacat 7334 7353 SEQ ID NO: 4225 atgtatcaaatggacattc 7685 7704 1 5 SEQ ID NO: 2888 ttaccaccagtttgtagat 7411 7430 SEQ ID NO: 4226 atctggaaccttgaagtaa 10739 10758 1 5 SEQ ID NO: 2889 ttgcagtgtatctggaaag 7548 7587 SEQ ID NO: 4227 cttttcacattagatgcaa 8420 8439 1 5 SEQ ID NO: 2890 cattcagcaggaacttcaa 7099 7718 SEQ ID NO: 4228 ttgaaggacttcaggaatg 12009 12028 1 5 SEQ ID NO: 2891 acacctgattttatagtcc 7958 7977 SEQ ID NO: 4229 ggactcaaggataacgtgt 12614 12633 1 5 SEQ ID NO: 2892 ggattccatcagttcagat 7992 8011 SEQ ID NO: 4230 atcttcaatgattatatcc 13124 13143 1 5 SEQ ID NO: 2893 ttgtagaaatgaaagtaaa 6112 8131 SEQ ID NO: 4231 tttatgattatgtcaacaa 12360 12379 1 5 SEQ ID NO: 2894 ctgaacagtgagctgcagt 8166 8175 SEQ ID NO: 4232 actggacttctctagtcag 8809 8828 1 5 SEQ ID NO: 2895 aatccaatctcctcttttc 8407 8426 SEQ ID NO: 4233 gaaaaatgaagtccggatt 11017 11036 1 5 SEQ ID NO: 2896 attttgattttcaagcaaa 8532 8551 SEQ ID NO: 4234 tttgcaagttaaagaaaat 14023 14042 1 5 SEQ ID NO: 2897 ttttgattttcaagcaaat 8533 8552 SEQ ID NO: 4235 atttgatttaagtgtaaaa 9622 9641 1 5 SEQ ID NO: 2898 tgattttcaagcaaatgca 8536 8555 SEQ ID NO: 4236 tgcaagttaaagaaaatca 14025 14044 1 5 SEQ ID NO: 2899 atgctgttttttggaaatg 8646 8864 SEQ ID NO: 4237 cattggtaggagacagcat 11203 11222 1 5 SEQ ID NO: 2900 tgctgttttttggaaatgc 8646 8665 SEQ ID NO: 4238 gcattggtaggagacagca 11202 11221 1 5 SEQ ID NO: 2901 aaaaaaatacactggagct 8706 8725 SEQ ID NO: 4239 agctagagggcctcttttt 10833 10852 1 5 SEQ ID NO: 2902 actggagcttagtaatgga 8716 8735 SEQ ID NO: 4240 tccactcacatcctccagt 1289 1308 1 5 SEQ ID NO: 2903 cttctggaaaagggtcatg 8886 8905 SEQ ID NO: 4241 catgaacccctacargaag 13759 13778 1 5 SEQ ID NO: 2904 ggaaaagggtcatggaaat 8891 8910 SEQ ID NO: 4242 atttgaaagttcgatttcc 9282 9301 1 5 SEQ ID NO: 2905 gggcctgccccagattctc 8910 8929 SEQ ID NO: 4243 gagaacattatggaggccc 9440 9459 1 5 SEQ ID NO: 2906 ttctcagctgagggaacac 8924 8943 SEQ ID NO: 4244 gtgtcttcaaagctgagaa 12416 12435 1 5 SEQ ID NO: 2907 gatgagggaacacatgaat 8930 8949 SEQ ID NO: 4245 attccagcttccccacatc 8338 8357 1 5 SEQ ID NO: 2908 vtttggactgtccaataag 8986 9005 SEQ ID NO: 4246 cttctgggatttcctaaag 11167 11186 1 5 SEQ ID NO: 2909 gcatccacaaacaatgaag 8260 9279 SEQ ID NO: 4247 cttactctgtcattgatgc 10227 10248 1 5 SEQ ID NO: 2910 cacaaacaatgaagggaat 8265 9284 SEQ ID NO: 4248 attccctgaagttgatgtg 11488 11507 1 5 SEQ ID NO: 2911 ccaaaatttctctgctgga 8415 9434 SEQ ID NO: 4249 tccatcacaaatccaaagg 9671 9690 1 5 SEQ ID NO: 2912 caaaatttctctgctggaa 8416 9435 SEQ ID NO: 4250 ttccatcacaaatcctttg 9670 9689 1 5 SEQ ID NO: 2913 tctgctggaaacaacgaga 8424 9444 SEQ ID NO: 4251 tctcaagagttacagcaga 13229 13248 1 5 SEQ ID NO: 2914 ctgctggaaacaacgagaa 9426 9445 SEQ ID NO: 4252 ttctcaagagttacagcag 13228 13247 1 5 SEQ ID NO: 2915 agaacattatggaggccca 9441 9480 SEQ ID NO: 4253 tgggcctgccccagattct 8909 8928 1 5 SEQ ID NO: 2916 agaagcaaatctggatttc 9475 9494 SEQ ID NO: 4254 gaaatcttcaatttattct 13821 13840 1 5 SEQ ID NO: 2917 tttctctctatgggaaaaa 9565 9564 SEQ ID NO: 4255 tttttgcaagttaaagaaa 14021 14040 1 5 SEQ ID NO: 2918 tcagagcatcaaatccttt 9712 9731 SEQ ID NO: 4256 aaagaaaatcaggatctga 14033 14052 1 5 SEQ ID NO: 2919 cagaaacaatgcattagat 9751 9770 SEQ ID NO: 4257 atctatgccatctcttctg 5633 5652 1 5 SEQ ID NO: 2920 tacacattaatcctgccat 10001 10020 SEQ ID NO: 4258 atggagtctttattgtgta 14089 14108 1 5 SEQ ID NO: 2921 agtcagatattgttgctca 10194 10213 SEQ ID NO: 4259 tgagaactacgagctgact 4807 4826 1 5 SEQ ID NO: 2922 ggagggyahycataacagt 10336 10355 SEQ ID NO: 4260 actggtggcaaaaccctcc 2734 2753 1 5 SEQ ID NO: 2923 caaaagccgaaattccaat 10404 10423 SEQ ID NO: 4261 attgaagtacctacttttg 8366 8385 1 5 SEQ ID NO: 2924 aaaagccgaaattccaatt 10405 10424 SEQ ID NO: 4262 aattgaagtacctactttt 8365 8384 1 5 SEQ ID NO: 2925 ttcaagcaagaacttaatg 10436 10455 SEQ ID NO: 4263 cattatggcccttcgtgaa 13258 13277 1 5 SEQ ID NO: 2926 cctcttacttttccattga 10578 105978 SEQ ID NO: 4264 tcaaaagaagcccaagagg 12947 12986 1 3 SEQ ID NO: 2927 tgaggccaacacttacttg 10663 10682 SEQ ID NO: 4265 caagcatctgattgactca 12678 12695 1 5 SEQ ID NO: 2928 cacttacttgaattccaag 10672 10891 SEQ ID NO: 4266 cttgaacacaaagtcagtg 6008 6027 1 5 SEQ ID NO: 2929 gaagtaaaagaaaattttg 10751 10770 SEQ ID NO: 4267 caaaaacattttcaacttc 5287 5306 1 5 SEQ ID NO: 2930 cctggaactctctccatgg 10882 10901 SEQ ID NO: 4268 ccatttacagatcttcagg 11372 11391 1 5 SEQ ID NO: 2931 agctggatgtaaccaccag 11184 11203 SEQ ID NO: 4269 ctggattccacatgcagct 11855 11874 1 5 SEQ ID NO: 2932 aaaattccctgaagttgat 11485 11504 SEQ ID NO: 4270 atcatatccgtgtaatttt 6765 6784 1 5 SEQ ID NO: 2933 cagatggcattgctgcttt 11613 11632 SEQ ID NO: 4271 aaagctgagaagaaatctg 12424 12443 1 5 SEQ ID NO: 2934 agatggcattgctgctttg 11614 11833 SEQ ID NO: 4272 caaagctgagaagaaatct 12423 12442 1 5 SEQ ID NO: 2935 tgttgaaacagtcctggat 11842 11861 SEQ ID NO: 4273 atccaagatgagatcaaca 13103 13122 1 5 SEQ ID NO: 2936 catattcaaaactgagttg 12229 12246 SEQ ID NO: 4274 caactctctgattactatg 13631 13650 1 5 SEQ ID NO: 2937 aaagatttatcaaaagaag 12938 12957 SEQ ID NO: 4275 cttcaatttattcttcttt 13826 13845 1 5 SEQ ID NO: 2938 attttccaactaatagaag 13034 13053 SEQ ID NO: 4276 cttcaaagacttaaaaaat 5014 8033 1 5 SEQ ID NO: 2939 aattatatccaagatgaga 13097 13116 SEQ ID NO: 4277 tctcttcctccatggaatt 10479 10498 1 5 SEQ ID NO: 2940 ttcaggaagcttctcaaga 13218 13237 SEQ ID NO: 4278 tcttcataagttcaatgaa 13183 13202 1 5 SEQ ID NO: 2941 ttgagcaatttctgcacag 13437 13456 SEQ ID NO: 4279 ctgttgaaagatttatcaa 12932 12951 1 5 SEQ ID NO: 2942 ctgatatacatcacggagt 13712 13731 SEQ ID NO: 4280 actcaatggtgaaattcag 7465 7484 1 5 SEQ ID NO: 2943 acatcacggagttactgaa 13719 13738 SEQ ID NO: 4281 ttcagaagctaagcaatgt 7239 7258 1 5 SEQ ID NO: 2944 actgcctatattgataaaa 13882 13901 SEQ ID NO: 4282 ttttggcaagctatacagt 8380 8399 1 5 SEQ ID NO: 2945 aggatggcattttttgcaa 14011 14030 SEQ ID NO: 4283 ttgcaagcaagtatttcct 3013 3032 1 5 SEQ ID NO: 2946 ttttttgcaagttaaagaa 14020 14039 SEQ ID NO: 4284 ttctctctatgggaaaaaa 9566 9585 1 5 SEQ ID NO: 2947 tccagaactcaagtcttca 1627 1646 SEQ ID NO: 4285 tgaaatgctgttttttgga 8641 8660 3 4 SEQ ID NO: 2948 agttagtgaaagaagttct 1956 1975 SEQ ID NO: 4286 agaatctgtaccaggaact 12564 12583 3 4 SEQ ID NO: 2949 atttacagctctgacaagt 5435 5454 SEQ ID NO: 4287 acttcagagaaatacaaat 11409 11428 3 4 SEQ ID NO: 2950 gattatctgaattcattca 6488 5507 SEQ ID NO: 4288 tgaaaccaatgacaaaatc 7429 7448 3 4 SEQ ID NO: 2951 gtgcccttctcggttgctg 26 45 SEQ ID NO: 4289 cagctgagcagacaggcac 6039 8058 2 4 SEQ ID NO: 2952 attcaagcacctccggaag 253 272 SEQ ID NO: 4290 cttcataagttcaatcaat 13184 13203 2 4 SEQ ID NO: 2953 gactgctgattcaagaagt 316 335 SEQ ID NO: 4291 acttcccaactctcaagtc 13415 13434 2 4 SEQ ID NO: 2954 ttgctgcagccatgtccag 483 502 SEQ ID NO: 4292 ctgggcagctgtatagcaa 5889 5908 2 4 SEQ ID NO: 2955 agaaagatgaacctactta 555 574 SEQ ID NO: 4293 taagtatgatttcaattct 10498 10517 2 4 SEQ ID NO: 2958 tgaagactctccaggaact 1095 1114 SEQ ID NO: 4294 agttcaatgaatttattca 13191 13210 2 4 SEQ ID NO: 2957 atctctcttgccacagctg 1210 1229 SEQ ID NO: 4265 cagcccagccatttgagat 9237 9256 2 4 SEQ ID NO: 2058 tctctcttgccacagctga 1211 1230 SEQ ID NO: 4296 tcagcccagccatttgaga 9236 9255 2 4 SEQ ID NO: 2959 tgaggtgtccagccccatc 1231 1250 SEQ ID NO: 4297 gatgggaaagccgccctca 5216 5235 2 4 SEQ ID NO: 2960 cchgaactcaagtcttcaa 1628 1647 SEQ ID NO: 4298 ttgaaagcagaacctctgg 5915 5934 2 4 SEQ ID NO: 2961 ctgaaaaagttagtgaaag 1949 1968 SEQ ID NO: 4299 ctttctcgggaatattcag 10631 10650 2 4 SEQ ID NO: 2962 tttttcccagacagtgtca 2246 2265 SEQ ID NO: 4300 tgacaggcattttgaaaaa 9730 9749 2 4 SEQ ID NO: 2963 ttttcccagacagtgtcaa 2247 2266 SEQ ID NO: 4301 ttgacaggcattttgaaaa 9729 9748 2 4 SEQ ID NO: 2964 cattcagaacaagaaaatt 3403 3422 SEQ ID NO: 4302 aattccaattttgagaatg 10414 10433 2 4 SEQ ID NO: 2965 tgaagagaagattgaattt 3628 3647 SEQ ID NO: 4303 aaatgtcagctcttgttca 10902 10921 2 4 SEQ ID NO: 2966 tttgaatggaacacaggca 3644 3653 SEQ ID NO: 4304 tgccagtttgaaaaaacaa 11815 11834 2 4 SEQ ID NO: 2967 ttctagattcgaatatcaa 4407 4420 SEQ ID NO: 4305 ttgacatgttaataaagaa 7377 7396 2 4 SEQ ID NO: 2968 gattcgaatatcaaattca 4412 4431 SEQ ID NO: 4306 tgaagtag5ccencasatc 7162 7181 2 4 SEQ ID NO: 2969 tgcaacgaccaacttgaag 5083 5102 SEQ ID NO: 4307 cttcaggttccatcgtgca 11384 11403 2 4 SEQ ID NO: 2970 ttaagctctcaaatgacat 5325 5344 SEQ ID NO: 4308 atgttgataaagaaattaa 7382 7401 2 4 SEQ ID NO: 2971 caatttaacaacaatgaat 6074 6093 SEQ ID NO: 4309 attcaaactgcctatattg 13876 13895 2 4 SEQ ID NO: 2972 tgaatacagccaggacttg 6088 6107 SEQ ID NO: 4310 caagagcacacggtcttca 10687 10706 2 4 SEQ ID NO: 2973 catcaatattgatcaattt 6421 6440 SEQ ID NO: 4311 aaattcccrgaagttgatg 11486 11505 2 4 SEQ ID NO: 2974 ttgagcatgtcaaacactt 7059 7078 SEQ ID NO: 4312 aagtaagtgctaggttcaa 9381 9400 2 4 SEQ ID NO: 2975 tgaaggagactattcagaa 7227 7246 SEQ ID NO: 4313 ttctgcacagaaatattca 13446 13465 2 4 SEQ ID NO: 2976 ttcaggctcttcagaaagc 7929 7948 SEQ ID NO: 4314 gcttgctaacctctctgaa 12312 12331 2 4 SEQ ID NO: 2977 tccacaaattgaacatccc 8787 8806 SEQ ID NO: 4315 gggacctaccaagagtgga 12533 12552 2 4 SEQ ID NO: 2978 tgaataccaatgctgaact 10167 10186 SEQ ID NO: 4316 agttcaatgaatttattca 13191 13210 2 4 SEQ ID NO: 2979 taaactaatagatgtaatc 12098 12917 SEQ ID NO: 4317 gattactatgaaaaattta 13840 13669 2 4 SEQ ID NO: 2980 ttgacctgtccattcaaaa 13680 13899 SEQ ID NO: 4318 ttttaaaagaaatcttcaa 13813 13832 2 4 SEQ ID NO: 2981 gggctgagtgcccttctcg 19 38 SEQ ID NO: 4319 cgaggccaggccgcagccc 84 103 1 4 SEQ ID NO: 2982 ggctgagtgcccttctcgg 20 39 SEQ ID NO: 4320 ccgaggccaggccgcagcc 83 102 1 4 SEQ ID NO: 2983 ctgagtgcccttctcggtt 22 41 SEQ ID NO: 4321 aaccgtgcctgaatctcag 11557 11578 1 4 SEQ ID NO: 2984 tctcggttgcrgccgctga 33 52 SEQ ID NO: 4322 tcagctgacctcatcgaga 2158 2187 1 4 SEQ ID NO: 2985 caggccgcagcccaggagc 90 109 SEQ ID NO: 4323 gctctgcagcttcatcctg 376 395 1 4 SEQ ID NO: 2986 gctggcgctgcctgcgctg 151 170 SEQ ID NO: 4324 cagcacagaccatttcagc 4252 4271 1 4 SEQ ID NO: 2987 tgctgctggcgggcgccag 177 196 SEQ ID NO: 4325 ctggatgtaaccaccagca 11186 11205 1 4 SEQ ID NO: 2988 ctggtctgtccaaaagatg 227 248 SEQ ID NO: 4326 catcctgaagaccagccag 388 407 1 4 SEQ ID NO: 2989 ctgagagttccagtggagt 291 310 SEQ ID NO: 4327 actcaccctggacattcag 3391 3410 1 4 SEQ ID NO: 2990 tccagtggagtccctggga 299 318 SEQ ID NO: 4328 tcccggagccaaggctgga 2683 2702 1 4 SEQ ID NO: 2991 aggttgagctggaggttcc 354 373 SEQ ID NO: 4329 ggaaccctctccctcacct 4735 4755 1 4 SEQ ID NO: 2992 tgagctggaggttccccag 358 377 SEQ ID NO: 4330 ctgggaggcatgatgctca 9171 9190 1 4 SEQ ID NO 2993 tctgcagcttcatcctgaa 378 397 SEQ ID NO: 4331 ttcaaatataatcgacaga 3269 3288 1 4 SEQ ID NO: 2994 gccagtgcaccctgaaaga 402 421 SEQ ID NO: 4332 tcttccgttctgtaatggc 5802 5821 1 4 SEQ ID NO: 2995 ctctgaggagtttgctgca 472 491 SEQ ID NO: 4333 tgcaagaatattttgagag 6348 6367 1 4 SEQ ID NO: 2996 aggtatgagctcaagctgg 500 519 SEQ ID NO: 4334 ccagtttccggggaaacct 12724 12743 1 4 SEQ ID NO: 2997 tcctttacccggagaaaga 543 562 SEQ ID NO: 4335 tctttttgggaagcaagga 2227 2248 1 4 SEQ ID NO: 2998 catcaagaggggcatcatt 582 602 SEQ ID NO: 4336 aatggtcaagttcctgatg 2285 2304 1 4 SEQ ID NO: 2999 tcctggttcccccagagac 609 628 SEQ ID NO: 4337 gtctctgaactcagaagga 13996 14015 1 4 SEQ ID NO: 3000 aagaagccaagcaagtgtt 630 649 SEQ ID NO: 4338 aacaaataaatggagtctt 14080 14099 1 4 SEQ ID NO: 3001 aagcaagtgttgtttctgg 638 657 SEQ ID NO: 4339 ccagagccaggtcgagctt 11050 11069 1 4 SEQ ID NO: 3002 tctggataccgtgtatgga 652 671 SEQ ID NO: 4340 tccatgtcccatttacaga 11364 1383 1 4 SEQ ID NO: 3003 ccactcactttaccgtcaa 678 697 SEQ ID NO: 4341 ttgattttaacaaaagtgg 6825 6644 1 4 SEQ ID NO: 3004 aggaagggcaatgtggcaa 701 720 SEQ ID NO: 4342 ttgcaagcaagtctttcct 3013 3032 1 4 SEQ ID NO: 3005 gcaatgtggcaacagaaat 708 727 SEQ ID NO: 4343 atttccataccccgtttgc 3488 3507 1 4 SEQ ID NO: 3006 caatgtggcaacagaaata 709 728 SEQ ID NO: 4344 tattcttcttttccaattg 13834 13863 1 4 SEQ ID NO: 3007 tggcaacagaaatatccac 714 733 SEQ ID NO: 4345 gtggcttcccatattgcca 1995 1914 1 4 SEQ ID NO: 3008 agagacctgggccagtgtg 737 756 SEQ ID NO: 4346 cacattacatttggtctct 2938 2957 1 4 SEQ ID NO: 3009 tgtgatcgcttcaagccca 752 771 SEQ ID NO: 4349 tgggaaagccgccctcaca 5218 5237 1 4 SEQ ID NO: 3010 gtgatcgcttcaagcccat 753 772 SEQ ID NO: 4350 atgggaaagccgccctcac 5217 5236 1 4 SEQ ID NO: 3011 cagcccacttgctctcatc 784 803 SEQ ID NO: 4351 gatgctgaacagtgagctg 8152 8171 1 4 SEQ ID NO: 3012 gctctcatcaaaggcatga 794 813 SEQ ID NO: 4352 tcataacagtactgtgacc 10345 10364 1 4 SEQ ID NO: 3013 ccttgtcaactctgatcag 819 838 SEQ ID NO: 4353 ctgagtgggtttatcaagg 12453 12472 1 4 SEQ ID NO: 3014 cttgtcaactctgatcagc 820 839 SEQ ID NO: 4354 gctgagtaggtttatcaag 12452 12471 1 4 SEQ ID NO: 3015 agccatctgcaaggagcaa 892 911 SEQ ID NO: 4355 ttgcaatgagctcatggct 3813 3832 1 4 SEQ ID NO: 3016 gccatctgcaaggagcaac 893 812 SEQ ID NO: 4356 gttgcaatgagctcatggc 3812 3831 1 4 SEQ ID NO: 3017 cttcctgcctttctcctac 816 835 SEQ ID NO: 4357 gtaggaataaatggagaag 9461 9480 1 4 SEQ ID NO: 3018 ctttctcctacaagaataa 924 943 SEQ ID NO: 4358 ttattgctgaatccaaaag 13658 13675 1 4 SEQ ID NO: 3019 gatcaacagccgcttcttt 997 1016 SEQ ID NO: 4359 aaagccatcactgatgatc 1669 1688 1 4 SEQ ID NO: 3020 atcaacagccgcttctttg 998 1017 SEQ ID NO: 4360 caaagccatcactgatgat 1668 1687 1 4 SEQ ID NO: 3021 acagccgcttctttggtga 1002 1021 SEQ ID NO: 4381 tcacaaatcctttggctgt 9675 9694 1 4 SEQ ID NO: 3022 aagatgggcctcgcatttg 1031 1050 SEQ ID NO: 4362 caaaatagaagggaatctt 2077 2096 1 4 SEQ ID NO: 3023 tgttttgaatactctccag 1090 1109 SEQ ID NO: 4383 ctggtaactactttaaaca 5495 5514 1 4 SEQ ID NO: 3024 ttgaagactctccaggaac 1094 1113 SEQ ID NO: 4364 gttcaatgaatttattcaa 13192 13211 1 4 SEQ ID NO: 3025 aactgaaaaaactaaccat 1110 1129 SEQ ID NO: 4365 atggcattttttgcaagtt 14014 14033 1 4 SEQ ID NO: 3026 ctgaaaaaactaaccatct 1112 1131 SEQ ID NO: 4386 agattgatgggcagttcag 4572 4591 1 4 SEQ ID NO: 3027 aaaactaaccatctctgag 1117 1138 SEQ ID NO: 4387 ctcaaagaatgactttttt 2578 2597 1 4 SEQ ID NO: 3028 tgagcaaaatatccagaga 1132 1151 SEQ ID NO: 4358 tctccagataaaaaactca 12209 12228 1 4 SEQ ID NO: 3029 caataagcthhttactgag 1182 1181 SEQ ID NO: 4369 ctcagatcaaagttaattg 12273 12292 1 4 SEQ ID NO: 3030 tactgagctgagaggcctc 1174 1193 SEQ ID NO: 4370 gagggtagtcataacagta 10337 10356 1 4 SEQ ID NO: 3031 gcctcagtgatgaagcagt 1188 1207 SEQ ID NO: 4371 actgttgactcaggaaggc 12580 12599 1 4 SEQ ID NO: 3032 agtcacatctctcttgcca 1204 1223 SEQ ID NO: 4372 tggccacatagcatggact 8866 8885 1 4 SEQ ID NO: 3033 atctctcttcccacagctg 1210 1229 SEQ ID NO: 4373 cagctgacctcatcgagat 2109 2188 1 4 SEQ ID NO: 3034 tctctcttgccacagctga 1211 1230 SEQ ID NO: 4374 tcagctgacctcatcgaga 2168 2187 1 4 SEQ ID NO: 3035 tgccacagctgattgaggt 1218 1237 SEQ ID NO: 4375 acctgcaccaaagctggca 13963 13982 1 4 SEQ ID NO: 3036 gccacagctgattgaggtg 1219 1238 SEQ ID NO: 4376 caccaaaaaccccaatggc 11248 11267 1 4 SEQ ID NO: 3037 tcactttacaagccttggt 1248 1267 SEQ ID NO: 4377 accatgctgaacagtgtga 8148 8167 1 4 SEQ ID NO: 3038 cccttctgatagatgtggt 1332 1351 SEQ ID NO: 4378 accacttacagctagaggg 10824 10843 1 4 SEQ ID NO: 3039 gtcacctacctggtggccc 1349 1368 SEQ ID NO: 4379 gggcgacctaagttgtgac 3439 3458 1 4 SEQ ID NO: 3040 ccttgtatgcgctgagcca 1440 1459 SEQ ID NO: 4380 tggctggtaacctaaaagg 5586 5605 1 4 SEQ ID NO: 3041 gacaaaccctacagggacc 1480 1499 SEQ ID NO: 4381 ggtcctttatgattatgtc 12355 12374 1 4 SEQ ID NO: 3042 tgctaattacctgatggaa 1516 1535 SEQ ID NO: 4382 ttcccaaaagcagtoogca 9938 9957 1 4 SEQ ID NO: 3043 tgactgcactggggatgaa 1546 1555 SEQ ID NO: 4383 ttcaggtccatgcaactca 10917 10938 1 4 SEQ ID NO: 3044 actgcactggggatgaaga 1557 1567 SEQ ID NO: 4384 tcttgaacacaaagtcagt 6007 8026 1 4 SEQ ID NO: 3045 atgaagattacacctattt 1560 1579 SEQ ID NO: 4385 aaatgaaagtaaagatcat 6116 8137 1 4 SEQ ID NO: 3046 accatggagcagttaactc 1610 1629 SEQ ID NO: 4386 gagtaaaccaaaacttggt 9024 9043 1 4 SEQ ID NO: 3047 gcagttaactccagaactc 1618 1637 SEQ ID NO: 4357 gagttactgaaaaagctgc 13727 13740 1 4 SEQ ID NO: 3048 cagaactcaagtcttcaat 1529 1545 SEQ ID NO: 4388 attggatatccaagatctg 1933 1952 1 4 SEQ ID NO: 3049 caggctctgcggaaaatgg 1703 1722 SEQ ID NO: 4389 ccatgaccrccagctcctg 2485 2504 1 4 SEQ ID NO: 3050 ccaggaggttcttcttcag 1738 1757 SEQ ID NO: 4393 ctgaaatacaatgctctgg 5519 5538 1 4 SEQ ID NO: 3051 ggttcttcttcagatttc 1744 1751 SEQ ID NO: 4391 gaaaaacttggaaacaacc 4439 4458 1 4 SEQ ID NO: 3052 tttccttgatgatgcttct 1758 1778 SEQ ID NO: 4392 agaatccagatacaagaaa 6893 6912 1 4 SEQ ID NO: 3053 ggagataagcgactggctg 1761 1900 SEQ ID NO: 4393 cagcatgcctagtttctcc 9952 9971 1 4 SEQ ID NO: 3054 gctgcctatcttatgttga 1796 1815 SEQ ID NO: 4394 tcaatatcaaaagcccage 12045 12064 1 4 SEQ ID NO: 3055 actttgtggcttcccatat 1890 1909 SEQ ID NO: 4395 atatctggaaccttgaagt 10737 10758 1 4 SEQ ID NO: 3056 gccaatatcttgaactcag 1910 1929 SEQ ID NO: 4396 ctgaactcagaaggatggc 14000 14019 1 4 SEQ ID NO: 3057 aatatcttgaactcagaag 1913 1932 SEQ ID NO: 4397 cttccattccgaatatatt 13378 13397 1 4 SEQ ID NO: 3058 ctcagaagaattggatatc 1924 1943 SEQ ID NO: 4398 gataaaagattactttgag 7273 7292 1 4 SEQ ID NO: 3059 aagaattggatatccaaga 1929 1948 SEQ ID NO: 4399 tcttcaatttattcttctt 13825 13844 1 4 SEQ ID NO: 3060 agaattggatatccaagat 1930 1949 SEQ ID NO: 4400 atcttsaatttattcttct 13824 13643 1 4 SEQ ID NO: 3061 tggatatvvaagatctgaa 1935 1954 SEQ ID NO: 4401 ttcacataccagaattcca 8325 8344 1 4 SEQ ID NO: 3062 atatccaagatctgaaaaa 1938 1957 SEQ ID NO: 4402 tttttaaccagtcagatat 10185 10204 1 4 SEQ ID NO: 3063 tatccaagatctgaaaaag 1939 1958 SEQ ID NO: 4403 ctttttaaccagtcagata 10184 10203 1 4 SEQ ID NO: 3064 caagatctgaaaaagttag 1943 1962 SEQ ID NO: 4404 ctaaattcccatggtcttg 4973 4992 1 4 SEQ ID NO: 3065 aagatctgaaaaagttagt 1944 1963 SEQ ID NO: 4405 actaaattcccatggtctt 4972 4991 1 4 SEQ ID NO: 3066 tgaaaaagttagtgaaaga 1650 1669 SEQ ID NO: 4406 tctttctcgggaatattca 10830 10649 1 4 SEQ ID NO: 3067 tccaactgtcatggacttc 1990 2009 SEQ ID NO: 4407 gaagcacatatgaactgga 13945 13964 1 4 SEQ ID NO: 3068 tcagaaaattctctcggaa 2007 2028 SEQ ID NO: 4408 ttcctttaacaattcctga 9501 9520 1 4 SEQ ID NO: 3069 ttccatcacttgacccagc 2052 2071 SEQ ID NO: 4409 gctgacatagggaatggaa 8441 8460 1 4 SEQ ID NO: 3070 cccagcctcagccaaaata 2065 2084 SEQ ID NO: 4410 tattctatccaagattggg 7820 7839 1 4 SEQ ID NO: 3071 agcctcagccaaaatagaa 2068 2087 SEQ ID NO: 4411 ttctatccaagattgggct 7822 7841 1 4 SEQ ID NO: 3072 atcttatatttgatccaaa 2091 2110 SEQ ID NO: 4412 tttgaaaaacaaagcagat 11621 11840 1 4 SEQ ID NO: 3073 tcttatatttgatccaaat 2092 2111 SEQ ID NO: 4413 attttttgcaagttaaaga 14019 14038 1 4 SEQ ID NO: 3074 cttcctaaagaaagcatgc 2117 2136 SEQ ID NO: 4414 gcatggcattatgatgaag 3614 3633 1 4 SEQ ID NO: 3075 ctaaagaaagcatgctgaa 2121 2140 SEQ ID NO: 4415 ttcagggtgtggagtttag 5894 5713 1 4 SEQ ID NO: 3076 taaagaaagcatgctgaaa 2122 2141 SEQ ID NO: 4416 tttcttaaacattccttta 9490 9509 1 4 SEQ ID NO: 3077 gagattggcttggaaggaa 2183 2202 SEQ ID NO: 4417 ttccctccattaagttctc 11709 11728 1 4 SEQ ID NO: 3078 ctttgagccaacattggaa 2208 2225 SEQ ID NO: 4418 ttccaatgaccaagaaaag 11068 11087 1 4 SEQ ID NO: 3079 cagacagtgtcaacaaagc 2253 2272 SEQ ID NO: 4419 gcttactggacgaactctg 6142 6161 1 4 SEQ ID NO: 3080 cagtgtcaacaaagctttg 2257 2275 SEQ ID NO: 4420 caaattcctggatatactg 9857 9878 1 4 SEQ ID NO: 3081 agtgtcaacaaagctttgt 2258 2277 SEQ ID NO: 4421 acaagaatacgtctacact 4359 4378 1 4 SEQ ID NO: 3082 ctgatggtgtctctaaggt 2299 2318 SEQ ID NO: 4422 acctcggaagaatcctcag 3333 3352 1 4 SEQ ID NO: 3083 tgatggtgtctctaaggtc 2299 2318 SEQ ID NO: 4423 gacctgcgcaacgagatca 8831 8850 1 4 SEQ ID NO: 3084 aaacatgagcaggatatgg 2351 2370 SEQ ID NO: 4424 ccatgatctacatttgttt 5796 8815 1 4 SEQ ID NO: 3085 gaagctgattaaagatttg 2395 2414 SEQ ID NO: 4425 caaaaacattttcaacttc 5267 5306 1 4 SEQ ID NO: 3086 aaagatttgaaatccaaag 2405 2424 SEQ ID NO: 4426 ctttaagttcagcatcttt 7614 7833 1 4 SEQ ID NO: 3087 gatgggtgcccgcactctg 2518 2537 SEQ ID NO: 4427 cagatttgaggattccatc 7983 8002 1 4 SEQ ID NO: 3088 gggatcccccagatgattg 2540 2559 SEQ ID NO: 4428 caatcacaagtcgattccc 9083 9102 1 4 SEQ ID NO: 3089 ttttcttcactacatcttc 2593 2812 SEQ ID NO: 4429 gaagtgtcagtggcaaaaa 10382 10401 1 4 SEQ ID NO: 3090 tcttcactacatcttcatg 2598 2615 SEQ ID NO: 4430 catggcattatgatgaaga 3615 3634 1 4 SEQ ID NO: 3091 tacatcttcatggagaatg 2603 2622 SEQ ID NO: 4431 cattatggaggcccatgta 9445 9464 1 4 SEQ ID NO: 3092 ttcatggagaatgcctttg 2809 2628 SEQ ID NO: 4432 caaaatcaactttaatgaa 6607 6628 1 4 SEQ ID NO: 3093 tcatggagaatgcctttga 2810 2629 SEQ ID NO: 4433 tcaacacaatcttcaatga 13116 13135 1 4 SEQ ID NO: 3094 tttgaactccccactggag 2624 2643 SEQ ID NO: 4434 ctccccaggacctttcaaa 9842 9881 1 4 SEQ ID NO: 3095 ttggaactcccactggagc 2625 2644 SEQ ID NO: 4435 gctccccaggacctttcaa 9841 9880 1 4 SEQ ID NO: 3096 tgaactccccactggagct 2626 2645 SEQ ID NO: 4436 agctccccaggacctttca 9840 8859 1 4 SEQ ID NO: 3097 cactggagctggcttcacg 2635 2654 SEQ ID NO: 4437 ctgtttctgagtcccagtg 9344 9383 1 4 SEQ ID NO: 3098 actggagctggattacagt 2636 2655 SEQ ID NO: 4438 actgtttctgagtcccagt 9343 9362 1 4 SEQ ID NO: 3099 agttgcaaatatcttcatc 2852 2871 SEQ ID NO: 4439 gatgataccaaaatacact 6599 6618 I 4 SEQ ID NO: 3100 gttgcaaatatcttcatct 2653 2672 SEQ ID NO: 4440 agatgatgccaaaatcaac 6698 6617 1 4 SEQ ID NO: 3101 aaatatcttcatctggagt 2658 2677 SEQ ID NO: 4441 actcagaaggatggcattt 14004 14023 1 4 SEQ ID NO: 3102 taaaactggaagtagccaa 2703 2722 SEQ ID NO: 4442 ttggttacaggaggcttta 7600 7619 1 4 SEQ ID NO: 3103 ggctgaactggtggcaaaa 2703 2722 SEQ ID NO: 4443 ttttcttttcagcccagcc 9228 9247 1 4 SEQ ID NO: 3104 tgtggagtttgtgacaaat 2758 2777 SEQ ID NO: 4444 attttcaagcaaatgcaca 8538 8557 1 4 SEQ ID NO: 3105 ttgtgacaaatatgggcat 2766 2785 SEQ ID NO: 4445 atgcgtctaccttacacaa 9521 9540 1 4 SEQ ID NO: 3108 atgaacaccaacttcttcc 2819 2838 SEQ ID NO: 4446 ggaagctgaagtttatcat 2877 2896 1 4 SEQ ID NO: 3107 cttccacgagtcgggtctg 2833 2852 SEQ ID NO: 4447 cagagctatcactgggaag 5235 5254 1 4 SEQ ID NO: 3108 gagtcgggtctggaggctc 2840 2856 SEQ ID NO: 4448 gagcttactggacgaactc 6140 6159 1 4 SEQ ID NO: 3109 cctaaaagctgggaagctg 2866 2885 SEQ ID NO: 4449 cagcctccccagccgtagg 12120 12139 1 4 SEQ ID NO: 3110 agctgggaagctgaagttt 2872 2891 SEQ ID NO: 4450 aaactgttaatttacagct 5463 5482 1 4 SEQ ID NO: 3111 ccagattagagctggaact 3114 3133 SEQ ID NO: 4451 agtttccggggaaacctgg 12726 12745 1 4 SEQ ID NO: 3112 ggataccctgaagtttgta 3208 3227 SEQ ID NO: 4452 tacagtattctgaaaatcc 8393 8412 1 4 SEQ ID NO: 3113 ctgaggctaccatgacatt 3252 3271 SEQ ID NO: 4453 aatgagctcatggcttcag 3817 3836 1 4 SEQ ID NO: 3114 tgtccagtgaagtccaaat 3297 3316 SEQ ID NO: 4454 attttgagaggaatcgaca 6357 6376 1 4 SEQ ID NO: 3115 aattccggattttgatgtt 3313 3332 SEQ ID NO: 4455 aacacatgaatcacaaatt 8938 8957 1 4 SEQ ID NO: 3116 ttccggattttgatgttga 3315 3334 SEQ ID NO: 4456 tcaaaacgagcttcaggaa 13207 13226 1 4 SEQ ID NO: 3117 cggaacaatcctcagagtt 3337 3351 SEQ ID NO: 4457 aacttgtacaactggtccg 4211 4230 1 4 SEQ ID NO: 3118 tcctcagagttaatgatga 3345 3364 SEQ ID NO: 4458 tcatcaattggttacagga 7593 7612 1 4 SEQ ID NO: 3119 ctcaccctggacattcaga 3392 3411 SEQ ID NO: 4459 tctgcagaacaatgctgag 12439 12458 1 4 SEQ ID NO: 3120 cattcagacaagaaaaatt 3403 3422 SEQ ID NO: 4461 aattgactttgtagaaatg 8104 8123 1 4 SEQ ID NO: 3121 actgaggtcgccctcatgg 3422 3441 SEQ ID NO: 4461 ccatgcaagtcagcccagt 10924 10943 1 4 SEQ ID NO: 3122 ttatttccataccccgttt 3488 3505 SEQ ID NO: 4462 aaactgcctatattgataa 13880 13599 1 4 SEQ ID NO: 3123 gtttgcaagcagaagccag 3501 3520 SEQ ID NO: 4463 ctggacttctcttcaaaac 5408 5427 I 4 SEQ ID NO: 3124 tttgcaagcagaagccaga 3502 3521 SEQ ID NO: 4464 tctgggtgtcgacagcaaa 5272 5291 1 4 SEQ ID NO: 3125 ttgcaagcagaagccagaa 3503 3522 SEQ ID NO: 4465 ttctgggtgtcgacagcaa 5271 6290 1 4 SEQ ID NO: 3126 ctgcttctccaaatggact 3554 3573 SEQ ID NO: 4466 agtcaagattgatgggcag 4567 4586 1 4 SEQ ID NO: 3127 tgctacacttatggctccc 3577 3596 SEQ ID NO: 4467 ggaggctttaagttcagca 7609 7628 1 4 SEQ ID NO: 3128 acagcttatggctccacag 3581 3600 SEQ ID NO: 4468 ctgtatagcaaattcctgt 5897 5916 1 4 SEQ ID NO: 3129 tttccaagagggtggcatg 3600 3819 SEQ ID NO: 4469 catggacttcttctggaaa 8877 8896 1 4 SEQ ID NO: 3130 ccaagagggtggcatggca 3603 3622 SEQ ID NO: 4470 tgcccagcaagcaagttga 9361 9380 1 4 SEQ ID NO: 3131 gtggcatggcattatgatg 3611 3630 SEQ ID NO: 4471 catccttaacaccttccac 8071 8090 1 4 SEQ ID NO: 3132 tgatgaagagaagattgaa 3625 3544 SEQ ID NO: 4472 ttcactgttcctgaaatca 7871 7890 1 4 SEQ ID NO: 3133 gaagagaagattgaatttg 3629 3848 SEQ ID NO: 4473 caaaaacattttcaacttc 6287 5308 1 4 SEQ ID NO: 3134 gagaagattgaatttgaat 3632 3551 SEQ ID NO: 4474 attcataatcccaactctc 8278 8297 1 4 SEQ ID NO: 3135 tttgaatggaacacaggca 3844 3853 SEQ ID NO: 4475 tgcctttgtgtacaccaaa 11238 11255 1 4 SEQ ID NO: 3136 aggcaccaatgtagatacc 3658 3577 SEQ ID NO: 4476 ggtaacctaaaaggagcct 5591 5610 1 4 SEQ ID NO: 3137 caaaaaaatgacttccaat 3676 3895 SEQ ID NO: 4477 attgaagtacctacttttg 8366 8385 1 4 SEQ ID NO: 3138 aaaaaaatgacttccaatt 3877 3698 SEQ ID NO: 4478 aattgaagtacctactttt 8385 8384 1 4 SEQ ID NO: 3139 aaaaaatgacttccaattt 3878 3697 SEQ ID NO: 4479 aaatccaatcacctctttt 8406 8425 1 4 SEQ ID NO: 3140 cagagtccctcaaaagaac 3780 3779 SEQ ID NO: 4480 gtctgtgggattccatctg 4090 4109 1 4 SEQ ID NO: 3141 aaattaatagttgcaatga 3603 3822 SEQ ID NO: 4481 tcataagttcaatgaattt 13186 13205 1 4 SEQ ID NO: 3142 ttcaacctccagaacatgg 3899 3918 SEQ ID NO: 4482 ccattgaccagatgctgaa 8142 8161 1 4 SEQ ID NO: 3143 tgggattgccagacttcca 3915 3934 SEQ ID NO: 4483 tggaaatgggcctgcccca 8003 8922 1 4 SEQ ID NO: 3144 cagtttgaaaattgagatt 3994 4013 SEQ ID NO: 4484 aatcacaactaataaactg 9541 9560 1 4 SEQ ID NO: 3145 gaaaattgagattcctttg 4000 4019 SEQ ID NO: 4485 caaaactaccacacatttc 13694 13713 1 4 SEQ ID NO: 3146 tttgccttttggtggcaaa 4015 4034 SEQ ID NO: 4486 tttgagaggaatcgacaaa 6359 6378 1 4 SEQ ID NO: 3147 ctccagagatctaaagatg 4036 4055 SEQ ID NO: 4487 catcaattggttacaggag 7504 7613 1 4 SEQ ID NO: 3148 tctaaagatgttagagact 4045 4064 SEQ ID NO: 4488 agtccttcatgtcctaaga 10033 10052 1 4 SEQ ID NO: 3149 ctgtgggattccatctgcc 4092 4111 SEQ ID NO: 4489 ggcatttgaaaaaaaacag 9735 9754 1 4 SEQ ID NO: 3150 atctgccatctcgagagtt 4104 4123 SEQ ID NO: 4490 aactttcaaaccctaagat 8568 8575 1 4 SEQ ID NO: 3151 tctcgagagttccaagtcc 4112 4131 SEQ ID NO: 4491 ggacattcctctagcgaga 8215 8234 1 4 SEQ ID NO: 3152 agtccctacttttaccatt 4126 4145 SEQ ID NO: 4492 aatgaatacagccaggact 8086 6105 1 4 SEQ ID NO: 3153 acttttaccattcccaagt 4133 4152 SEQ ID NO: 4493 actttgtagaaatgaaagt 8109 8128 1 4 SEQ ID NO: 3154 cattcccaagttgtatcaa 4141 4160 SEQ ID NO: 4494 ttgaaggacttcaggaatg 12009 12028 1 4 SEQ ID NO: 3155 accacatgaaggctgactc 4284 4303 SEQ ID NO: 4495 gagtaaaccaaaacttggt 9024 9043 1 4 SEQ ID NO: 3156 tttcctacaatgtgcaagg 4317 4338 SEQ ID NO: 4496 cctttaacaattcctgaaa 9503 9522 1 4 SEQ ID NO: 3157 ctggagaaacaacatatga 4338 4357 SEQ ID NO: 4497 tcattctgggtctttccag 11035 11054 1 4 SEQ ID NO: 3158 atcatgtgatgggtctcta 4378 4397 SEQ ID NO: 4498 tagaattacagaaaatgat 5585 6584 1 4 SEQ ID NO: 3159 catgtgatgggtctctacg 4380 4399 SEQ ID NO: 4499 cgtaggcaccgtgggcatg 12133 12152 1 4 SEQ ID NO: 3160 ttctagattcgaatatcaa 4407 4426 SEQ ID NO: 4500 ttgatgatgctgtcaagaa 7308 7327 1 4 SEQ ID NO: 3161 tggggaccacagatgtctg 4499 4515 SEQ ID NO: 4501 cagaattccagacttccca 8334 8363 1 4 SEQ ID NO: 3162 ctaacactggccggctcaa 4644 4663 SEQ ID NO: 4502 ttgaggctattgatgttag 5984 7003 1 4 SEQ ID NO: 3163 taacactggccggctcaat 4845 4664 SEQ ID NO: 4503 attgaggctattgatgtta 6983 7002 1 4 SEQ ID NO: 3164 aacactggccggctcaatg 4646 4665 SEQ ID NO: 4504 cattgaggctattgatgtt 6982 7001 1 4 SEQ ID NO: 3165 ctggccggctcaatggaga 4850 4669 SEQ ID NO: 4505 tctccatctgcgctaccag 12073 12092 1 4 SEQ ID NO: 3166 agataacaggaagatatga 4713 4732 SEQ ID NO: 4506 tcatctcctttattcatct 10210 10229 1 4 SEQ ID NO: 3167 tccctcacctccacctctg 4745 4764 SEQ ID NO: 4507 cagatatatatctcaggga 8184 8203 1 4 SEQ ID NO: 3168 agctgactttaaaatctga 4818 4837 SEQ ID NO: 4508 tcaggctcttcagaaagct 7930 7949 1 4 SEQ ID NO: 3169 ctgactttaaaatctgaca 4820 4839 SEQ ID NO: 4309 tgtcaagataaacaatcag 8740 8759 1 4 SEQ ID NO: 3170 caagatggatatgaccttc 4873 4592 SEQ ID NO: 4510 gaagtagtactgcatcttg 6843 6862 1 4 SEQ ID NO: 3171 gctgcgttctgaatatcag 4909 4928 SEQ ID NO: 4511 ctgagtcccagtgcccagc 9350 9369 1 4 SEQ ID NO: 3172 cgttctgaatatcaggctg 4913 4932 SEQ ID NO: 4512 cagcaagtacctgagaacg 8611 8630 1 4 SEQ ID NO: 3173 aattcccatggtcttgagt 4976 4993 SEQ ID NO: 4513 actcagatcaaagtttatt 12272 12291 1 4 SEQ ID NO: 3174 tggtcttgagttaaatgct 4984 5003 SEQ ID NO: 4514 agcacagtacgaaaaacca 10609 10828 1 4 SEQ ID NO: 3175 cttgagttaaatgctgaca 4988 5007 SEQ ID NO: 4515 tgtccctagaaatctcaag 10042 10061 1 4 SEQ ID NO: 3176 ttgagttaaatgctgacat 4989 6008 SEQ ID NO: 4516 atgtccctagaaatctcaa 10041 10060 1 4 SEG ID NO: 3177 tgagttaaatgctgacatc 4990 5009 SEQ ID NO: 4517 gatggaaccctctaaatca 4733 4752 1 4 SEQ ID NO: 3178 acttgaagtgtagtctcct 5094 5113 SEQ ID NO: 4518 aggaaactcagatcaaagt 12267 12285 1 4 SEQ ID NO: 3179 agtgtagtctcctggtgct 5100 5119 SEQ ID NO: 4519 agcagccagtggcaccact 12514 12533 1 4 SEQ ID NO: 3180 gtgctggagaatgagctga 5114 5133 SEQ ID NO: 4520 tcagccaggtttatagcac 7734 7753 1 4 SEQ ID NO: 3181 ctggggcatctatgaaatt 5151 5170 SEQ ID NO: 4521 aatttctgattaccaccag 13579 13698 1 4 SEQ ID NO: 3182 atggccgcttcagggaaca 5178 5197 SEQ ID NO: 4522 tgttttttggaaatgccat 8849 8668 1 4 SEQ ID NO: 3183 ttcagtctggatgggaaag 5207 5228 SEQ ID NO: 4523 ctttgacaggcattttgaa 9727 9746 1 4 SEQ ID NO: 3184 ccatgattctgggtgtcga 5265 5284 SEQ ID NO: 4524 tcgatgcacatacaaatgg 5838 5857 1 4 SEQ ID NO: 3185 aaaacattttcaacttcaa 5289 5308 SEQ ID NO: 4825 ttgatgttagagtgcttttt 6993 7012 1 4 SEQ ID NO: 3186 cttaagctctcaaatgaca 6324 5343 SEQ ID NO: 4526 tgtcctacaacaagttaag 7265 7274 1 4 SEQ ID NO: 3187 ttaagctctcaaatgacat 5325 5344 SEQ ID NO: 4627 atgtcctacaacaagttaa 7264 7273 1 4 SEQ ID NO: 3188 catgatgggctcatatgct 5341 5380 SEQ ID NO: 4528 agcatctttggctcacatg 7824 7843 1 4 SEQ ID NO: 3189 tgggctcatatgctgaaat 5346 5355 SEQ ID NO: 4529 atttatcaaaagaagccca 12942 12961 1 4 SEQ ID NO: 3100 actggacttctcttcaaaa 5407 6426 SEQ ID NO: 4530 ttttggcaagctatacagt 8350 8399 1 4 SEQ ID NO: 3191 acttctcttcaaaacttga 5412 5431 SEQ ID NO: 4531 tcaattgggagagacaagt 6604 8523 1 4 SEQ ID NO: 3192 ctgacaagttttataagca 5445 5464 SEQ ID NO: 4532 tgctttgtgagtttatcag 9693 9712 1 4 SEQ ID NO: 3193 aagttttataagcaaactg 5450 6469 SEQ ID NO: 4533 cagtcatgtagaaaaactt 4429 4448 1 4 SEQ ID NO: 3194 ctattaatttacagctaca 5466 5485 SEQ ID NO: 4534 tgtactggaaaacgtacag 6388 6407 1 4 SEQ ID NO: 3195 ttacagctacagccctatt 5474 5493 SEQ ID NO: 4635 aatattgatcaatttgtaa 8425 6444 1 4 SEQ ID NO: 3196 tctggtaactactttaaac 5494 5513 SEQ ID No: 4636 gtttgaaaaacaaagcaga 11820 11839 1 4 SEQ ID NO: 3197 tttaaacagtgacctgaaa 5506 5525 SEQ ID NO: 4337 tttcatttgaaagaataaa 7032 7051 1 4 SEQ ID NO: 3198 ttaaacagtgacctagggt 5507 5526 SEQ ID NO: 4638 atttcaagcaagaacttaa 10434 10453 1 4 SEQ ID NO: 3199 cagtgacctgaaatacaat 5512 5531 SEQ ID NO: 4539 attggcgtggagcttactg 6131 6150 1 4 SEQ ID NO: 3200 tgtggctggtaacctaaaa 5584 5603 SEQ ID NO: 4540 ttttgctggagaagccaca 10765 10784 1 4 SEQ ID NO: 3201 ttatcagcaagctataaag 5657 5676 SEQ ID NO: 4541 ctttgcactatgttcataa 12784 12783 1 4 SEQ ID NO: 3202 ggttcagggtgtggagttt 5692 5711 SEQ ID NO: 4542 aaacacctaagagtaaacc 9014 9033 1 4 SEQ ID NO: 3203 attcagactcactgcattt 5775 5794 SEQ ID NO: 4543 aaatgctgacatagggaat 8437 8458 1 4 SEQ ID NO: 3204 ttcagactcactgcatttc 5776 5796 SEQ ID NO: 4544 gaaatattatgaacttgaa 13312 13331 1 4 SEQ ID NO: 3205 tacaaatggcaatgggaaa 5648 5867 SEQ ID NO: 4545 tttcctaaagctggatgta 11178 11195 1 4 SEQ ID NO: 3206 gctgtatagcaaattcctg 5898 5915 SEQ ID NO: 4646 caggtccatgcaagtcagc 10919 10938 1 4 SEQ ID NO: 3207 tgagcagacaggcacctgg 6043 6062 SEQ ID NO: 4547 ccagcttccccacatctca 8341 8360 1 4 SEQ ID NO: 3208 ggcacctggaaactcaaga 6053 6072 SEQ ID NO: 4548 tcttcgtgtttcaactgcc 11221 11240 1 4 SEQ ID NO: 3209 tgaatacagccaggacttg 6088 6107 SEQ ID NO: 4549 caagtaagtgctaggttca 9380 9399 1 4 SEQ ID NO: 3210 gaatacagccaggacttgg 6089 6108 SEQ ID NO: 4550 ccaacacttacttgaattc 10668 10687 1 4 SEQ ID NO: 3211 ctggacgaactctggctga 6147 6466 SEQ ID NO: 4551 tcagaaagctaccttccag 7939 7958 1 4 SEQ ID NO: 3212 ttttactcagtgagcccat 6201 6220 SEQ ID NO: 4552 atggacttcttctggaaaa 8878 8897 1 4 SEQ ID NO: 3213 gatgagagatgvvgttgag 6241 6260 SEQ ID NO: 4553 ctcatctcctttctttatc 10209 10228 1 4 SEQ ID NO: 3214 aattgttgctttagtaaag 6277 6298 SEQ ID NO: 4554 cttttctaaacttgaaatt 9064 9083 1 4 SEQ ID NO: 3215 cttttctaaagtatgataa 6285 6304 SEQ ID NO: 4555 ttatgaacttgaagaaaag 13318 13337 1 4 SEQ ID NO: 3216 tttgtaaagtatgatgggg 6287 6308 SEQ ID NO: 4556 ttttcacattagatgcaaa 8421 8440 1 4 SEQ ID NO: 3217 tccattaacctcccatttt 6320 6339 SEQ ID NO: 4557 aaaattgatgatatctgga 10727 10746 1 4 SEQ ID NO: 3218 ccattaacctcccattttt 6321 8340 SEQ ID NO: 4558 aaaagggtcatggaaatgg 8893 8912 1 4 SEQ ID NO: 3219 agaatattttgagaggaat 6346 6365 SEQ ID NO: 4559 ctcaatttttgatttcaag 8528 5647 1 4 SEQ ID NO: 3220 agaata1111gagaggaat 6352 6371 SEQ ID NO: 4560 attccctccattaagttct 11708 11727 1 4 SEQ ID NO: 3221 attatagttgtactggaaa 6380 6399 SEQ ID NO: 4561 tttcaagcaagaacttaat 10435 104 54 1 4 SEQ ID NO: 3222 gaagcacatcaatattgat 6415 6434 SEQ ID NO: 4562 atcagttcagataaacttc 7999 8018 1 4 SEQ ID NO: 3223 acatcaatattgatcaatt 6420 6439 SEQ ID NO: 4563 aattccctgaagttgatgt 11487 11506 1 4 SEQ ID NO: 3224 gaaaactcccacagcaagc 6465 6484 SEQ ID NO: 4564 gctttctcttccacatttc 10060 10079 1 4 SEQ ID NO: 3225 ctgaattcattcaattggg 6494 6513 SEQ ID NO: 4565 cccatttacagatcttcag 11371 11390 1 4 SEQ ID NO: 3226 tgaattcattcaattggga 6495 6514 SEQ ID NO: 4566 tcccatttacagatcttca 11370 11389 1 4 SEQ ID NO: 3227 aactgactgctctcacaaa 6540 6559 SEQ ID NO: 4567 tttgaggattccatcagtt 7987 8006 1 4 SEQ ID NO: 3228 acaagtatagaattacagc 6558 6577 SEQ ID NO: 4568 tctggctccctcaactttt 9050 9069 1 4 SEQ ID NO: 3229 atcaactttaatgaaaaac 6611 6630 SEQ ID NO: 4569 gtttattgaaaatattgat 6811 6830 1 4 SEQ ID NO: 3230 tgatttgaaaatagctatt 6694 6713 SEQ ID NO: 4570 aatattattgatgaaatca 6716 6735 1 4 SEQ ID NO: 3231 atttgaaaatagctattgc 6696 6715 SEQ ID NO: 4571 gcaagaacttaatggaaat 10441 10460 1 4 SEQ ID NO: 3232 attgctaatattattgatg 6710 6729 SEQ ID NO: 4572 catcacactgaataccaat 10159 10178 1 4 SEQ ID NO: 3233 gaaaaattaaaaagtcttg 6737 6756 SEQ ID NO: 4573 caagagcttatgggatttc 11161 11180 1 4 SEQ ID NO: 3234 actatcatatccgtgtaat 6762 6781 SEQ ID NO: 4574 attactttgagaaattagt 7281 7300 1 4 SEQ ID NO: 3235 tattgattttaacaaaagt 6823 6842 SEQ ID NO: 4575 acttgacttcagagaaata 11404 11423 1 4 SEQ ID NO: 3236 ctgcagcagcttaagagac 6914 6933 SEQ ID NO: 4576 gtcttcagtgaagctgcag 10599 10718 1 4 SEQ ID NO: 3237 aaaacaacacattgaggct 6973 6992 SEQ ID NO: 4577 agcctcacctcttactttt 10571 10590 1 4 SEQ ID NO: 3238 ttgagcatgtcaaacactt 7059 7078 SEQ ID NO: 4578 aagtagctgagaaaatcaa 7104 7123 1 4 SEQ ID NO: 3239 tttgaagtagctgagaaaa 7100 7119 SEQ ID NO: 4579 ttttcacattagatgcaaa 8421 8440 1 4 SEQ ID NO: 3240 ttagtagagttggcccacc 7199 7218 SEQ ID NO: 4580 ggtggactcttgctgctaa 7776 7795 1 4 SEQ ID NO: 3241 tgaaggagactattcagaa 7227 7246 SEQ ID NO: 4581 ttctcaattttgattttca 8526 6545 1 4 SEQ ID NO: 3242 gagactattcagcagctaa 7232 7251 SEQ ID NO: 4582 ttagccacagctctgtctc 10301 10320 1 4 SEQ ID NO: 3243 aattagttggatttattga 7293 7312 SEQ ID NO: 4583 tcaagaagcttaatgaatt 7320 7339 1 4 SEQ ID NO: 3244 gcttaatgaattatctttt 7327 7346 SEQ ID NO: 4584 aaaacgagcttcaggacgc 13209 13228 1 4 SEQ ID NO: 3245 ttaacaaattccttgacat 7365 7384 SEQ ID NO: 4585 atgtcctacaacaagttaa 7254 7273 1 4 SEQ ID NO: 3246 aaattaaagtcatttgatt 7394 7413 SEQ ID NO: 4586 aatcctttgacaggcattt 9723 9742 1 4 SEQ ID NO: 3247 gactcaatggtgaaattca 7464 7483 SEQ ID NO: 4587 tgaaattcaatcacaagtc 9076 9095 1 4 SEQ ID NO: 3248 gaaattcaggctctggaac 7475 7494 SEQ ID NO: 4588 gttctcaattttgattttc 8525 8544 1 4 SEQ ID NO: 3249 actaccacaaaaagctgaa 7492 7511 SEQ ID NO: 4589 ttcaggaactattgctagt 10645 10664 1 4 SEQ ID NO: 3250 ccaaaataaccttaatcat 7578 7597 SEQ ID NO: 4590 atgatttccctgaccttgg 10950 10969 1 4 SEQ ID NO: 3251 aaataaccttaatcatcaa 7581 7600 SEQ ID NO: 4591 ttgaagtaaaagaaaattt 10749 10766 1 4 SEQ ID NO: 3252 tttaagttcagcatctttg 7615 7634 SEQ ID NO: 4592 caaatctggatttcttaaa 9480 9499 1 4 SEQ ID NO: 3253 caggtttatagcacacttg 7739 7758 SEQ ID NO: 4593 caagggttcactgttcctg 7865 7884 1 4 SEQ ID NO: 3254 gttcactgttcctgaaatc 7870 7889 SEQ ID NO: 4594 gattctcagatgagggaac 8922 8941 1 4 SEQ ID NO: 3255 cactgttcctgaaatcaag 7873 7892 SEQ ID NO: 4595 cttgaacacaaagtcagtg 6008 6027 1 4 SEQ ID NO: 3256 actgttcctgaaatcaaga 7874 7893 SEQ ID NO: 4596 tcttgaacacaaagtcagt 6007 6026 1 4 SEQ ID NO: 3257 gcctgcctttgaagtcagt 7909 7928 SEQ ID NO: 4597 actgttgactcaggaaggc 12580 12599 1 4 SEQ ID NO: 3258 taacagatttgaggattcc 7980 7999 SEQ ID NO: 4598 ggaagcttctcaagagtta 13222 13241 1 4 SEQ ID NO: 3259 gttttccacaccagaattt 8050 8069 SEQ ID NO: 4599 aaattcctctgctggaaac 9418 9437 1 4 SEQ ID NO: 3260 tcagaaccattgaccagat 8136 8155 SEQ ID NO: 4600 atctgcagaacaatgctga 12438 12457 1 4 SEQ ID NO: 3261 tagcgagaatcaccctgcc 8226 8245 SEQ ID NO: 4601 ggcagcttctggcttgcta 12301 12320 1 4 SEQ ID NO: 3262 ccttaatgattttcaagtt 8299 8318 SEQ ID NO: 4602 aactgttgactcaggaagg 12579 12598 1 4 SEQ ID NO: 3263 acataccagaattccagct 8328 8347 SEQ ID NO: 4603 agctgccagtccttcatgt 10026 10045 1 4 SEQ ID NO: 3264 aatgctgacatagggaatg 8438 8457 SEQ ID NO: 4604 cattaatcctgccatcatt 10005 10024 1 4 SEQ ID NO: 3265 atgctgacatagggaatgg 8439 8458 SEQ ID NO: 4605 ccatttgagatcacggcat 9245 9264 1 4 SEQ ID NO: 3266 aaccacctcagcaaacgaa 8458 8477 SEQ ID NO: 4606 ttcgttttccattaaggtt 9291 9310 1 4 SEQ ID NO: 3267 agcaggtatcgcagcttcc 8476 8495 SEQ ID NO: 4607 ggaagtggccctgcatgct 10972 10991 1 4 SEQ ID NO: 3268 tgcacaactctcaaaccct 8551 8570 SEQ ID NO: 4608 agggaaagagaagattgca 13501 13520 1 4 SEQ ID NO: 3269 aggagtcagtgaagttctc 8592 8611 SEQ ID NO: 4609 gagaacttactatcatcct 13788 13807 1 4 SEQ ID NO: 3270 tttttggaaatgccattga 8652 8671 SEQ ID NO: 4610 tcaatgaatttattcaaaa 13194 13213 1 4 SEQ ID NO: 3271 aatggagtgattgtcaaga 8729 8748 SEQ ID NO: 4611 tcttttcagcccagccatt 9231 9250 1 4 SEQ ID NO: 3272 gtcaagataaacaaccagc 8741 8760 SEQ ID NO: 4612 gctgactttaaaatctgac 4819 4838 1 4 SEQ ID NO: 3273 tccacaaattgaacatccc 8787 8806 SEQ ID NO: 4613 gggatttcctaaagctgga 11172 11191 1 4 SEQ ID NO: 3274 ttgaacatccccaaactgg 8795 8814 SEQ ID NO: 4614 ccagtttccagggactcaa 12603 12622 1 4 SEQ ID NO: 3275 acatccccaaactggactt 8799 8818 SEQ ID NO: 4615 aagtcgattcccagcatgt 9090 9109 1 4 SEQ ID NO: 3276 acttctctagtcaggctga 8814 8833 SEQ ID NO: 46l6 tcagatggaaaaatgaagt 11010 11029 1 4 SEQ ID NO: 3277 tgaatcacaaattagttcc 8944 8963 SEQ ID NO: 4617 gaaagtccataatggttca 12817 12836 1 4 SEQ ID NO: 3278 agaaggacccctcacttcc 8968 8987 SEQ ID NO: 46l8 ggaagaagaggcagcttct 12292 12311 1 4 SEQ ID NO: 3279 ttggactgtccaataagat 8988 9007 SEQ ID NO: 4619 tctaaatgcagtagccaa 11634 11653 1 4 SEQ ID NO: 3280 actgtccaataagatcaat 8992 9011 SEQ ID NO: 4620 attgataaaaccatacagt 13891 13910 1 4 SEQ ID NO: 3281 ctgtccaataagatcaata 8993 9012 SEQ ID NO: 4621 tattgataaaaccatacag 13890 13909 1 4 SEQ ID NO: 3262 gtttatgaatctggctccc 9041 9060 SEQ ID NO: 4622 gggaatctgatgaggaaac 12255 12274 1 4 SEQ ID NO: 3283 atgaatctggctccctcaa 9045 9064 SEQ ID NO: 4623 ttgagttgcccaccatcat 11667 11686 1 4 SEQ ID NO: 3284 ctcaacttttctaaacttg 9059 9078 SEQ ID NO: 4624 caagatcgcagactttgag 11653 11672 1 4 SEQ ID NO: 3285 ctaaaggcatggcactgct 9129 9148 SEQ ID NO: 4625 aacagaaacaatgcattag 9749 9768 1 4 SEQ ID NO: 3286 aaggcatggcactgtttgg 9132 9151 SEQ ID NO: 4626 ccaagaaaaggcacacctc 11077 11096 1 4 SEQ ID NO: 3287 atccacaaacaatgaaggg 9262 9281 SEQ ID NO: 4627 ccctaacagatttgaggat 7977 7996 1 4 SEQ ID NO: 3288 ggaatttgaaagttcgttt 9279 9298 SEQ ID NO: 4628 aaacaaacacaggcattcc 9655 9674 1 4 SEQ ID NO: 3289 aataactatgcactgtttc 9332 9351 SEQ ID NO: 4629 gaaatactgttttcctatt 12836 12855 1 4 SEQ ID NO: 3290 gaaacaacgagaacattat 9432 9451 SEQ ID NO: 4630 ataaactgcaagatttttc 13608 13627 1 4 SEQ ID NO: 3291 ttcttgaaaacgacaaagc 9599 9618 SEQ ID NO: 4631 gctttccaatgaccaagaa 11085 11084 1 4 SEQ ID NO: 3292 ataagaaaaacaaacacag 9648 9667 SEQ ID NO: 4632 ctgtgctttgtgagtttat 9690 9709 1 4 SEQ ID NO: 3293 aaaacaaacacaggcattc 9654 9673 SEQ ID NO: 4633 gaatttgaaagttcgtttt 9280 9299 1 4 SEQ ID NO: 3294 gcattccatcacaaatcct 9667 9686 SEQ ID NO: 4634 aggaagtggccctgaatgc 10971 10990 1 4 SEQ ID NO: 3295 tctgaaaaaaacagaaaca 9740 9759 SEQ ID NO: 4635 tgttgaaagatttatcaaa 12933 12952 1 4 SEQ ID NO: 3296 caatgcattagattttgtc 9757 9776 SEQ ID NO: 4636 gacaagaaaaaggggattg 10279 10298 1 4 SEQ ID NO: 3297 caaagctgaaaaatctcag 9817 9836 SEQ ID NO: 4637 ctgagaacttcatcatttg 11438 11457 1 4 SEQ ID NO: 3298 cctggatacactgttccag 9863 9882 SEQ ID NO: 4638 ctggacttctctagtcagg 8810 8829 1 4 SEQ ID NO: 3299 gttgaagtgtctccattca 9890 9909 SEQ ID NO: 4639 tgaatctggctccctcaac 9046 9065 1 4 SEQ ID NO: 3300 tttctccatcctaggttct 9964 9983 SEQ ID NO: 4640 agaatccagatacaagaaa 6893 6912 1 4 SEQ ID NO: 3301 ttctccatcctaggttctg 9965 9984 SEQ ID NO: 4641 cagaatccagatacaagaa 6892 6911 1 4 SEQ ID NO: 3302 tcattagagctgccagtcc 10019 10038 SEQ ID NO: 4642 ggacagtgaaatattatga 13305 13324 1 4 SEQ ID NO: 3303 tgctgaactttttaaccag 10177 10196 SEQ ID NO: 4643 ctggatgtaaccaccagca 11186 11205 1 4 SEQ ID NO: 3304 ctcctttcttcatcttcgt 10214 10233 SEQ ID NO: 4644 atgaagcttgctccaggag 13772 13791 1 4 SEQ ID NO: 3305 tgtcattgatgcactgcag 10234 10253 SEQ ID NO: 4645 ctgcgctaccagaaagaca 12080 12099 1 4 SEQ ID NO: 3306 tgatgcactgcagtacaaa 10240 10259 SEQ ID NO: 4646 tttgagttgcccaccatca 11666 11685 1 4 SEQ ID NO: 3307 agctctgtctctgagcaac 10309 10328 SEQ ID NO: 4647 gttgaccacaagcttagct 10547 10566 1 4 SEQ ID NO: 3308 agccgaaattccaattttg 10408 10427 SEQ ID NO: 4648 caaagctggcaccagggct 13971 13990 1 4 SEQ ID NO: 3309 ttgagaatgaatttcaagc 10424 10443 SEQ ID NO: 4649 gcttcaggaagcttctcaa 13216 13235 1 4 SEQ ID NO: 3310 aaacctactgtctcttcct 10469 10488 SEQ ID NO: 4650 aggaaggccaagccagttt 12591 12610 1 4 SEQ ID NO: 3311 tacttttccattgagtcat 10583 10602 SEQ ID NO: 4651 atgattatgtcaacaagta 12363 12382 1 4 SEQ ID NO: 3312 tcaggtccctgcaagtcag 10918 10937 SEQ ID NO: 4652 ctgacatcttaggcactga 5001 6020 1 4 SEQ ID NO: 3313 atgcaagtcagcccagttc 10926 10945 SEQ ID NO: 4653 gaactcagaaggatggcat 14002 14021 1 4 SEQ ID NO: 3314 tgaatgctaacactaagaa 10983 11002 SEQ ID NO: 4654 ttctcaattttgattttca 8526 8545 1 4 SEQ ID NO: 3315 agaagatcagatggaaaaa 11004 11023 SEQ ID NO: 4655 ttttctaaatggaacttct 12173 12192 1 4 SEQ ID NO: 3316 ggctattcattctccatcc 11264 11283 SEQ ID NO: 4656 ggatctaaatgcagtagcc 11632 11651 1 4 SEQ ID NO: 3317 aaagttttggctgataaat 11288 11307 SEQ ID NO: 4657 atttcttaaacattccttt 9489 9508 1 4 SEQ ID NO: 3318 agttttggctgataaattc 11290 11309 SEQ ID NO: 4658 gaatctggctccctcaact 9047 9086 1 4 SEQ ID NO: 3319 ccgggctgaaactaaatga 11316 11335 SEQ ID NO: 4659 tcattctgggtctttccag 11035 11054 1 4 SEQ ID NO: 3320 cagagaaatacaaatctat 11413 11432 SEQ ID NO: 4660 atagcatggacttcttctg 8873 8892 1 4 SEQ ID NO: 3321 gaggtaaaattccctgaag 11480 11499 SEQ ID NO: 4662 cttctggcttgctaacctc 12306 12325 1 4 SEQ ID NO: 3322 cttttttgagataaccgtg 11545 11564 SEQ ID NO: 4663 cacggagttactgaaaaag 13723 13742 1 4 SEQ ID NO: 3323 gctggaattgtcattcctt 11735 11754 SEQ ID NO: 4664 aaggcatctccacctcagc 12102 12121 1 4 SEQ ID NO: 3324 gtgtataatgccacttgga 11795 11814 SEQ ID NO: 4665 tccaagatgagatcaacac 13104 13123 1 4 SEQ ID NO: 3325 attccacatgcagctcaac 11859 11878 SEQ ID NO: 4666 gttgagaagccccaagaat 6254 6273 1 4 SEQ ID NO: 3326 tgaagaagatggcaaattt 11992 12011 SEQ ID NO: 4667 aaattcccttttcctttca 9220 9239 1 4 SEQ ID NO: 3327 atcaaaagcccagcgttca 12050 12069 SEQ ID NO: 4668 tgaaagtcaagcatctgat 12669 12688 1 4 SEQ ID NO: 3328 gtgggcatggatatggatg 12143 12162 SEQ ID NO: 4669 catccttaacaccttccac 8071 8090 1 4 SEQ ID NO: 3329 aaatggaacttctactaca 12179 12198 SEQ ID NO: 4670 Cgtaccataagccatattc 10088 10107 1 4 SEQ ID NO: 3330 aaaaactcaccatattcaa 12219 12238 SEQ ID NO: 4671 ttgatgttagagtgctttt 6993 7012 1 4 SEQ ID NO: 3331 ctgagaagaaatctgcaga 12428 12447 SEQ ID NO: 4672 tctgcccagaaatattcag 13447 13466 1 4 SEQ ID NO: 3332 acaatgctgagtgggttta 12447 12466 SEQ ID NO: 4673 taaatggagtctttattgt 14086 14105 1 4 SEQ ID NO: 3333 caatgctgagtgggtttat 12448 12467 SEQ ID NO: 4674 ataaatggagtctttattg 14085 14104 1 4 SEQ ID NO: 3334 ttaggcaaattgatgatat 12477 12496 SEQ ID NO: 4675 atattgtcagtgcctctaa 13392 13411 1 4 SEQ ID NO: 3335 ataaactaacagatgtaat 12897 12916 SEQ ID NO: 4676 attactatgaaaaatttat 13641 13660 1 4 SEQ ID NO: 3336 ccaactaatagaagataac 13039 13058 SEQ ID NO: 4677 gttattttgctaaacttgg 14052 14071 1 4 SEQ ID NO: 3337 ttaattatatccaagatga 13095 13114 SEQ ID NO: 4678 tcatcctctaattttttaa 13800 13819 1 4 SEQ ID NO: 3338 tttaaattgttgaaagaaa 13151 13170 SEQ ID NO: 4679 tttcatttgaaagaataaa 7032 7051 1 4 SEQ ID NO: 3339 aagttcaatgaatttattc 13190 13209 SEQ ID NO: 4680 gaataccaatgctgaactt 10168 10187 1 4 SEQ ID NO: 3340 ttgaagaaaagatagtccg 13326 13345 SEQ ID NO: 4681 ctgagagaagtgtcttcaa 12407 12426 1 4 SEQ ID NO: 3341 acttccattctgaatatat 13377 13396 SEQ ID NO: 4682 atatctggaaccttgaagt 10737 10756 1 4 SEQ ID NO: 3342 cacagaaatattcaggaat 13451 13470 SEQ ID NO: 4683 attccctgaagttgatgtg 11488 11507 1 4 SEQ ID NO: 3343 ccattgcgacgaagaaaat 13560 13579 SEQ ID NO: 4684 atttttattcctgccatgg 10103 10122 1 4 SEQ ID NO: 3344 tataaactgcaagattttt 13607 13626 SEQ ID NO: 4685 aaaattcaaactgcctata 13873 13892 1 A SEQ ID NO: 3345 tctgattactatgaaaaat 13637 13656 SEQ ID NO: 4686 atttgtaagaaaatacaga 6436 6455 1 4 SEQ ID NO: 3346 ggagttactgaaaaagctg 13726 13745 SEQ ID NO: 4687 cagcatgcctagtttctcc 9952 9971 1 1 SEQ ID NO: 3347 tgaagcttgctccaggaga 13773 13792 SEQ ID NO: 4688 tctcctttcttcatcttca 10213 10232 1 4 SEQ ID NO: 3348 tgaactggacctgcaccaa 13955 13974 SEQ ID NO: 4689 ttggtcgagcaagggttca 7856 7875 1 4 SEQ ID NO: 3349 ttgctaaacttgggggagg 14058 14077 SEQ ID NO: 4690 cctcctacagtggtggcaa 4230 4249 1 4 SEQ ID NO: 3350 gattcgaatatcaaattca 4412 4431 SEQ ID NO: 4691 tgaaaacgacaaagcaatc 9603 8622 3 3 SEQ ID NO: 3351 atttgtttgtcaaagaagt 4551 4570 SEQ ID NO: 4692 acttttctaaacttgaaat 9063 9082 3 3 SEQ ID NO: 3352 tctcggttgctgccgctga 33 52 SEQ ID NO: 4693 tcagcccagccatttgaga 9236 9255 2 3 SEQ ID NO: 3353 gctgaggagcccgcccagc 47 66 SEQ ID NO: 4694 gctggatgtaaccaccagc 11185 11204 2 3 SEQ ID NO: 3354 ctggtctgtccaaaagatg 227 246 SEQ ID NO: 4695 catcagaaccattgaccag 8134 8153 2 SEQ ID NO: 3355 ctgagagttccagtggagt 291 310 SEQ ID NO: 4696 actcaatggtgaaattcag 7465 74 84 2 3 SEQ ID NO: 3356 cagtgcaccccgaaagagg 404 423 SEQ ID NO: 4697 cctcacttcctttggactg 8977 8996 2 SEQ ID NO: 3357 ctctgaggagtttgctgca 472 491 SEQ ID NO: 4698 tgcaaacttgacttcagag 11399 11418 2 SEQ ID NO: 3358 acatcaagaggggcatcat 582 601 SEQ ID NO: 4699 atgacgttcttgagcatgt 7050 7089 2 3 SEQ ID NO: 3359 ctgatcagcagcagccagt 830 849 SEQ ID NO: 4700 actggacttctctagtcag 8809 8828 2 7 SEQ ID NO: 3360 ggacgctaagaggaagcat 865 884 SEQ ID NO: 4701 atgcctacgttccatgtcc 11354 11373 2 3 SEQ ID NO: 3361 agctgttttgaagactctc 1087 1106 SEQ ID NO: 4702 gagaagtgtcttcaaagct 12411 12430 2 3 SEQ ID NO: 3362 tgaaaaaactaaccatctc 1113 1132 SEQ ID NO: 4703 gagatcaacacaatcttca 13112 13131 2 3 SEQ ID NO: 3363 ctgagctgagaggcctcag 1176 1195 SEQ ID NO: 4704 ctgaattactgcacctcag 3035 3054 2 3 SEQ ID NO: 3364 tgaaacgtgtgcatgccaa 1311 1330 SEQ ID NO: 4705 ttggtagagcaagggttca 7856 7875 2 3 SEQ ID NO: 3365 ccttgtatgcgctgagcca 1440 1459 SEQ ID NO: 4706 tggcactgtttggagaagg 9138 9157 2 3 SEQ ID NO: 3366 aggagctgctggacattgc 1500 1519 SEQ ID NO: 4707 gcaagtcagcccagttcct 10928 10947 2 3 SEQ ID NO: 3367 atttgattctgcgggtcat 1575 1594 SEQ ID NO: 4708 atgaaaccaatgacaaaat 7428 7447 2 3 SEQ ID NO: 3368 tccagaactcaagtcttca 1627 1646 SEQ ID NO: 4709 tgaaatacaatgctctgga 5520 5539 2 3 SEQ ID NO: 3369 ggttcttcttcagactttc 1744 1763 SEQ ID NO: 4710 gaaataccaagtcaaaacc 10455 104 74 2 3 SEQ ID NO: 3370 gttgatgaggagtccttca 1810 1829 SEQ ID NO: 4711 Cgaaaaagctgcaatcaac 13734 13753 2 3 SEQ ID NO: 3371 tccaagatctgaaaaagtt 1941 1960 SEQ ID NO: 4712 aactgcttctccaaatgga 3552 3371 2 3 SEQ ID NO: 3372 agttagtgaaagaagttct 1956 1975 SEQ ID NO: 4713 agaattcataatcccaact 8275 82 94 2 3 SEQ ID NO: 3373 gaagggaatcttatatttg 2084 2103 SEQ ID NO: 4714 caaaacctactgtctcttc 10467 10488 2 3 SEQ ID NO: 3374 ggaagctctttttgggaag 2221 2240 SEQ ID NO: 4715 cttcacataccagaattcc 8324 8343 2 3 SEQ ID NO: 3375 tggaataatgctcagtgtt 2374 2393 SEQ ID NO: 4716 aacaaacacaggcattcca 9656 9675 2 3 SEQ ID NO: 3376 gatttgaaatccaaagaag 2408 2427 SEQ ID NO: 4717 cttcatgtccctagaaatc 10037 10056 2 3 SEQ ID NO: 3377 tccaaagaagtcccggaag 2417 2436 SEQ ID NO: 4118 cttcagcctgctttctgga 4951 4970 2 3 SEQ ID NO: 3378 aggaagggctcaaagaatg 2570 2589 SEQ ID NO: 4719 cattagagctgccagtcct 10020 10039 2 3 SEQ ID NO: 3379 agaatgacttttttcttca 2583 2602 SEQ ID NO: 4720 tgaagatgacgacttttct 12160 12179 2 3 SEQ ID NO: 3380 tttgtgacaaatatgggca 2765 2784 SEQ ID NO: 4721 tgccagtttgaaaaacaaa 11815 11834 2 3 SEQ ID NO: 3381 ctgaggctaccatgacatt 3252 3271 SEQ ID NO: 4722 aatgtcagctcttgttcag 10903 10922 2 3 SEQ ID NO: 3382 gtagataccaaaaaaatga 3668 3687 SEQ ID NO: 4723 tcatttgccctcaacctac 11450 11469 2 3 SEQ ID NO: 3383 aaatgacttccaatttccc 3681 3700 SEQ ID NO: 4724 gggaactgttgaaagattc 1292 7 12946 2 3 SEQ ID NO: 3384 atgacttccaatttccctg 3683 3702 SEQ ID NO: 4725 caggagaacttactatcat 13785 13804 2 3 SEQ ID NO: 3385 atctgccatctcgagagtt 4104 4123 SEQ ID NO: 4726 aactcctccactgaaagat 9547 9566 2 3 SEQ ID NO: 3386 atttgtttgtcaaagaagt 4551 4570 SEQ ID NO: 4727 acttccgtttaccagaaat 8247 8266 2 3 SEQ ID NO: 3387 gcagagcttggcctctctg 5135 5154 SEQ ID NO: 4728 cagagctttctgccactgc 13518 13537 2 3 SEQ ID NO: 3388 atatgctgaaatgaaattt 5353 5372 SEQ ID NO: 4729 aaattcaaactgcctatat 13874 13893 2 3 SEQ ID NO: 3389 tcaaaacttgacaacattt 5420 5439 SEQ ID NO: 4730 aaatacttccacaaattga 8780 8799 2 3 SEQ ID NO: 3390 cagtgacctgaaatacaat 5512 5531 SEQ ID NO: 473l attgaacatccccaaactg 8794 8813 2 3 SEQ ID NO: 3391 tacaaatggcaatgggaaa 5848 5867 SEQ ID NO: 4732 tttcaactgcctttgtgta 11229 11248 2 3 SEQ ID NO: 3392 cttttgtaaagtatgataa 6285 6304 SEQ ID NO: 4733 ttattgctgaatccaaaag 13656 13675 2 3 SEQ ID NO: 3393 ttgtaaagtatgataaaaa 6288 6307 SEQ ID NO: 4734 ttttcaagcaaatgcacaa 8539 8558 2 3 SEQ ID NO: 3394 tccattaacctcccatttt 6320 6339 SEQ ID NO: 4735 aaaagaaaattttgctgga 10756 10775 2 3 SEQ ID NO: 3395 gattatctgaattcattca 6488 6507 SEQ ID NO: 4736 tgaagtagaccaacaaatc 7162 7181 2 3 SEQ ID NO: 3396 aattgggagagacaagttt 6506 6525 SEQ ID NO: 4737 aaactaaatgatctaaatt 11324 11343 2 3 SEQ ID NO: 3397 atttgacaatagctattgc 6696 6715 SEQ ID NO: 4738 gcaatttctgcacagaaat 13441 13460 2 3 SEQ ID NO: 3398 tgagcatgtcaaacacttt 7060 7079 SEQ ID NO: 4739 aaagccattcagtctctca 12971 12990 2 3 SEQ ID NO: 3399 ttgaagatgttaacaaatt 7356 7375 SEQ ID NO: 4740 aattccatatgaaagtcaa 12660 12679 2 3 SEQ ID NO: 3400 acttgtcacctacatttct 7753 7772 SEQ ID NO: 4741 agaatattttgatccaagt 13276 13295 2 3 SEQ ID NO: 3401 gttttccacaccagaattt 8050 8069 SEQ ID NO: 4742 aaatctggatttcttaaac 9481 9500 2 3 SEQ ID NO: 3402 ataagtacaaccaaaattt 9405 9424 SEQ ID NO: 4743 aaataaatggagtctttat 14083 14102 2 3 SEQ ID NO: 3403 cgggacctgcggggctgag 8 27 SEQ ID NO: 4744 ctcagttaactgtgtcccg 11571 11590 1 3 SEQ ID NO: 3404 agtgcccttctcggttgct 25 44 SEQ ID NO: 4745 agcatctgattgactcact 12678 12697 1 3 SEQ ID NO: 3405 gctgaggagcccgcccagc 47 66 SEQ ID NO: 4746 gctgattgaggtgtccagc 1225 1244 1 3 SEQ ID NO: 3406 gagcagcccgcccagccag 50 69 SEQ ID NO: 4747 ctggatcacagagtccctc 3752 3771 1 3 SEQ ID NO: 3407 gggccgcgaggccgaggcc 72 91 SEQ ID NO: 4748 ggccctgatccccgagccc 1363 13 82 1 3 SEQ ID NO: 3408 ccaggccgcagcccaggag 89 108 SEQ ID NO: 4749 ctcccggagccaaggctgg 2682 2701 1 3 SEQ ID NO: 3409 ggagccgccccaccgcagc 104 123 SEQ ID NO: 4750 gctgttttgaagactctcc 1088 1107 1 3 SEQ ID NO: 3410 gaagaggaaatgctggaaa 200 219 SEQ ID NO: 4751 tttcaagttcctgaccttc 8309 8328 1 3 SEQ ID NO: 3411 caaaagatgcgacccgatt 237 256 SEQ ID NO: 4752 aatcttattggggattttg 7085 7104 1 3 SEQ ID NO: 3412 cttcaagcacctccggaag 253 272 SEQ ID NO: 4753 cttccacatttcaaggaat 10067 10086 1 3 SEQ ID NO: 3413 gttccagtggagtccctgg 297 316 SEQ ID NO: 4754 ccagcaagtacctgagaac 8810 8629 1 3 SEQ ID NO: 3414 gactgctgattcaagaagt 316 335 SEQ ID NO: 4755 acttgaagaaaagatagtc 13324 13343 1 3 SEQ ID NO: 3415 gtgccaccaggatcaactg 333 352 SEQ ID NO: 4756 cagtgaagctgcagggcac 10704 10723 1 3 SEQ ID NO: 3416 gatcaactgcaaggttgag 343 362 SEQ ID NO: 4757 ctcacctccacctctgatc 4748 4767 1 3 SEQ ID NO: 3417 actgcaaggttgagctgga 348 367 SEQ ID NO: 4758 tccactcacatcctccagt 1289 1308 1 3 SEQ ID NO: 3418 ccagctctgcagcttcatc 373 392 SEQ ID NO: 4759 gatgtggtcacctacctgg 1343 1362 1 3 SEQ ID NO: 3419 agcttcatcctgaagccca 383 402 SEQ ID NO: 4760 tggtgctggagaatgagct 5112 5131 1 3 SEQ ID NO: 3420 cttcatcctgaagaccagc 385 404 SEQ ID NO: 4761 gctggagtaaaactggaag 2696 2715 1 3 SEQ ID NO: 3421 ccagccagtgcaccctgaa 399 418 SEQ ID NO: 4762 ttcaagatgactgcactgg 1539 1558 1 3 SEQ ID NO: 3422 cagtgcaccctgaaagagg 404 423 SEQ ID NO: 4763 cctcacagagctatcactg 5230 5249 1 3 SEQ ID NO: 3423 tggcttcaaccctgagggc 427 446 SEQ ID NO: 4764 gcccactggtcgcctgcca 3533 3552 1 3 SEQ ID NO: 3424 cttcaaccctgagggcaaa 430 449 SEQ ID NO: 4765 tttgagccaacattggaag 2207 2226 1 3 SEQ ID NO: 3425 ttcaaccctgagggcaaag 431 450 SEQ ID NO: 4766 ctttgacaggcattttgaa 9727 9746 1 3 SEQ ID NO: 3426 cttgctgaagaaaaccaag 451 470 SEQ ID NO: 4767 cttgaaattcaatcacaag 9074 9093 1 3 SEQ ID NO: 3427 tgctgaagaaaaccaagaa 453 472 SEQ ID NO: 4768 ttctgctgccttatcagca 5647 5666 1 3 SEQ ID NO: 3428 ttgctgcagccatgtccag 483 502 SEQ ID NO: 4769 ctggtcagtttgcaagcaa 3004 3023 1 3 SEQ ID NO: 3429 tgctgcagccatgtccagg 484 503 SEQ ID NO: 4770 cctggtcagtttgcaagca 3003 3022 1 3 SEQ ID NO: 3430 agccatgtccaggtatgag 490 509 SEQ ID NO: 4771 ctcacatcctccagtggct 1293 1312 1 3 SEQ ID NO: 3431 agctcaagctggccattcc 507 526 SEQ ID NO: 4772 ggaactaccacaaaaagct 7489 7508 1 3 SEQ ID NO: 3432 agaagggaagcaggttttc 526 545 SEQ ID NO: 4773 gaaatcttcaatttattct 13821 13840 1 3 SEQ ID NO: 3433 cagggaagcaggttttcct 528 547 SEQ ID NO: 4774 aggacaccaaaataacctc 7572 7591 1 3 SEQ ID NO: 3434 agaaagatgaacctactta 555 574 SEQ ID NO: 4775 taagaactttgccacttct 4852 4871 1 3 SEQ ID NO: 3435 atcctgaacatcaagaggg 575 594 SEQ ID NO: 4776 ccctaacagatttgaggat 7977 7996 1 3 SEQ ID NO: 3436 tcctgaacatcaagagggg 576 595 SEQ ID NO: 4777 cccctaacagatttgagga 7976 7995 1 3 SEQ ID NO: 3437 ctgaacatcaagaggggca 578 597 SEQ ID NO: 4778 tgcctgcctttgaagtcag 7908 7927 1 3 SEQ ID NO: 3438 aacatcaagcggggcatca 581 600 SEQ ID NO: 4779 tgataaaaaccaagatgtt 6298 6317 1 3 SEQ ID NO: 3439 acatcaagaggggcatcat 582 601 SEQ ID NO: 4780 atgataaaaaccaagatgt 6297 S316 1 3 SEQ ID NO: 3440 tcatttctgccctcctggt 597 616 SEQ ID NO: 4781 accaccagtttgtagatga 7413 7432 1 3 SEQ ID NO: 3441 ttcccccagagacagaaga 615 634 SEQ ID NO: 4782 tcttccacatttcaaggaa 10066 10085 1 3 SEQ ID NO: 3442 gaagaagccaagcaagtgt 629 648 SEQ ID NO: 4783 acaccttccacattccttc 8079 8098 1 3 SEQ ID NO: 3443 ttgtttctggataccgtgt 647 666 SEQ ID NO: 4784 acactaaatacttccacaa 8775 8794 1 3 SEQ ID NO: 3444 tgtatggaaactgctccac 663 682 SEQ ID NO: 4785 gtggaggcaacacattaca 2928 2947 1 3 SEQ ID NO: 3445 aaactgctccactcacttt 670 689 SEQ ID NO: 4786 aaagaaacagcatttgttt 4540 4559 1 3 SEQ ID NO: 3446 actcactttaccgtcaaga 680 699 SEQ ID NO: 4787 tcttacttttccattgagt 10580 10599 1 3 SEQ ID NO: 3447 ctttaccgtcaagacgagg 685 704 SEQ ID NO: 4788 cctccagctcctgggaaag 2491 2510 1 3 SEQ ID NO: 3448 ttaccgtcaagacgaggaa 687 706 SEQ ID NO: 4789 ttcctaaagctggatgtaa 11177 11196 1 3 SEQ ID NO: 3449 acgaggaagggcaatgtgg 698 717 SEQ ID NO: 4790 ccacaagtcatcatctcgt 5964 5983 1 3 SEQ ID NO: 3450 cgaggaagggcaatgtggc 699 718 SEQ ID NO: 4791 gccagaagtgagatcctcg 3515 3534 1 3 SEQ ID NO: 3451 gaggaagggcaatgtggca 700 719 SEQ ID NO: 4792 tgccagtctccatgacctc 2476 2495 1 3 SEQ ID NO: 3452 ggaagggccatgtggcaac 702 721 SEQ ID NO: 4793 gttgctcttaaggacttcc 13364 13383 1 3 SEQ ID NO: 3453 gaagggcaatgtggcaaca 703 722 SEQ ID NO: 4794 tgttgatgaggagtccttc 1809 1828 1 3 SEQ ID NO: 3454 caggcatcagcccacttgc 777 796 SEQ ID NO: 4795 gcaagtctttcctggcctg 3019 3038 1 3 SEQ ID NO: 3455 aggcatcagcccacttgct 778 797 SEQ ID NO: 4796 agcaagtctttcctggcct 3018 3037 1 3 SEQ ID NO: 3456 tcagcccacttgctctcat 783 802 SEQ ID NO: 4797 atgaaagtcaagcatctga 12668 12687 1 3 SEQ ID NO: 3457 gtcaactctgatcagcagc 823 842 SEQ ID NO: 4798 gctgactttaaaatctgac 4819 4838 1 3 SEQ ID NO: 3458 ggacgctaagaggaagcat 865 884 SEQ ID NO: 4799 atgcactgtttctgagtcc 933 9 9358 1 3 SEQ ID NO: 3459 aaggagcaacacctcttcc 902 921 SEQ ID NO: 4800 ggaatatcttagcatcctt 13465 13484 1 3 SEQ ID NO: 3460 aggagcaacacctcttcct 903 922 SEQ ID NO: 4801 aggaatatcttagcatcct 13464 13483 1 3 SEQ ID NO: 3461 caacacctcttcctgcctt 908 927 SEQ ID NO: 4802 aaggctgactctgtggttg 4 29 2 4311 1 3 SEQ ID NO: 3462 aacacctcttcctgccttt 909 928 SEQ ID NO: 4803 aaagcaggccgaagctgtt 1075 1094 1 3 SEQ ID NO: 3463 acaagaataagtatgggat 933 952 SEQ ID NO: 4804 atccatgatctacatttgt 6794 6813 1 3 SEQ ID NO: 3464 caagaataagtatgggatg 934 953 SEQ ID NO: 4805 catcactttacaagccttg 1246 1265 1 3 SEQ ID NO: 3465 tagcacaagtgacacagac 954 973 SEQ ID NO: 4806 gtctcttcgttctatgcta 4592 4611 1 3 SEQ ID NO: 3466 agcacaagtgacacagact 955 974 SEQ ID NO: 4807 agtctcttcgttctatgct 4591 4610 1 3 SEQ ID NO: 3467 gcacaagtgacacagacct 956 975 SEQ ID NO: 4808 aagtgtagtctcccggtgc 5099 5118 1 3 SEQ ID NO: 3468 aacttgaagacacaccaaa 978 997 SEQ ID NO: 4809 tttgaggattccatcagtt 7987 8006 1 3 SEQ ID NO: 3469 gcttctttggtgaaggtac 1008 1027 SEQ ID NO: 4810 gtacctacttttggcaagc 8372 8391 1 3 SEQ ID NO: 3470 ctttggtgaaggtactaag 1012 1031 SEQ ID NO: 4811 cttatgggatttcctaaag 11167 11186 1 3 SEQ ID NO: 3471 tactaagaagatgggcccc 1024 1043 SEQ ID NO: 4812 gagggtagtcataacagta 10337 10356 1 3 SEQ ID NO: 3472 tttgagagcaccaaatcca 1046 1065 SEQ ID NO: 4813 tggaagtgtcagtggcaaa 10380 10399 1 3 SEQ ID NO: 3473 agagcaccaaatccacatc 1050 1069 SEQ ID NO: 4814 gatggatatgaccttctct 4876 4895 1 3 SEQ ID NO: 3474 agctgttttgaagactctc 1087 1106 SEQ ID NO: 4815 gagaacatactgggcagct 5880 5899 1 3 SEQ ID NO: 3475 tgaaaaaactaaccatctc 1113 1132 SEQ ID NO: 4816 gagaaaatcaatgccttca 7112 7131 1 3 SEQ ID NO: 347t gaaaaaactaaccatctct 1114 1133 SEQ ID NO: 4817 agagccaggtcgagctttc 11052 11071 1 3 SEQ ID NO: 3477 tctgagcaaaatatccaga 1130 1149 SEQ ID NO: 4818 1ctgatgaggaaactcaga 12260 12279 1 3 SEQ ID NO: 3478 tctcttcaataagctggtt 1156 1175 SEQ ID NO: 4819 aacctcccattttttgaga 6326 6345 1 3 SEQ ID NO: 3479 ctgagctgagaggcctcag 1176 1195 SEQ ID NO: 4820 ctgatccccgagccctcag 1367 1386 1 3 SEQ ID NO: 3480 tgaagcagtcacatctctc 1198 1217 SEQ ID NO: 4821 gagaaaatcaatgccttca 72 7131 1 3 SEQ ID NO: 3481 aagcagtcacatctctctt 1200 1219 SEQ ID NO: 4822 aagaggcagcttctggctt 12297 12316 1 3 SEQ ID NO: 3482 ctctcttgccacagctgat 1212 1231 SEQ ID NO: 4823 atcaaaagaagcccaagag 1294 6 12965 1 3 SEQ ID NO: 3483 tcttgccacagctgattga 1215 1234 SEQ ID NO: 4824 tcaaagttaattgggaaga 12279 12298 1 3 SEQ ID NO: 3484 cttgccacagctgattgag 1216 1235 SEQ ID NO: 4825 ctcaattttgattttcaag 8528 8547 1 3 SEQ ID NO: 3485 tgaggtgtccagccccatc 1231 1250 SEQ ID NO: 4826 gatggaaccctctccctca 4733 4752 1 3 SEQ ID NO: 3486 tcagtgtggacagcctcag 1267 1286 SEQ ID NO: 4827 ctgacatcttaggcactga 5001 6020 1 3 SEQ ID NO: 3487 acatcctccagtggctgaa 1296 1315 SEQ ID NO: 4828 ttcagaagctaagcaatgt 7239 7258 1 3 SEQ ID NO: 3488 gcacagcagctgcgagaga 1385 1404 SEQ ID NO: 4829 tctctgaaagacaacgtgc 12323 12342 1 3 SEQ ID NO: 3489 cagcagctgcgagagatct 1388 1407 SEQ ID NO: 4830 agataacattaaacagctg 13051 13070 1 3 SEQ ID NO: 3490 gcgagggatcagcgcagcc 1415 1434 SEQ ID NO: 4831 ggctcaacacagacatcgc 5718 5737 1 3 SEQ ID NO: 3491 aagacaaaccctacaggga 1478 1497 SEQ ID NO: 4832 tcccagaaaacctcttctt 3936 3955 1 3 SEQ ID NO: 3492 caggagctgctggacattg 1499 1518 SEQ ID NO: 4833 caatggagagtccaacctg 4860 4679 1 3 SEQ ID NO: 3493 aggagctgctggacattgc 1500 1519 SEQ ID NO: 4834 gcaagggttcactgttcct 7864 7883 1 3 SEQ ID NO: 3494 ctgctggacattgctaatt 1505 1524 SEQ ID NO: 4935 aattgggaagaagaggcag 12287 12306 1 3 SEQ ID NO: 3495 gattacacctatttgattc 1565 1584 SEQ ID NO: 4836 gaatattttgagaggaatc 6353 6372 1 3 SEQ ID NO: 3496 atttgattctgcgggtcat 1575 1594 SEQ ID NO: 4837 atgaagtagaccaacaaat 7161 7180 1 3 SEQ ID NO: 3497 tctgcgggtcattggaaat 1582 1601 SEQ ID NO: 4838 atttgtaagaaaatacaga 6436 6455 1 3 SEQ ID NO: 3498 aaccatggagcagttaact 1609 1628 SEQ ID NO: 4839 agtttctccatcctaggtt 9962 9981 1 3 SEQ ID NO: 3499 ggagcagttaactccagaa 1615 1634 SEQ ID NO: 4840 ttctgaaaatccaatctcc 8400 8419 1 3 SEQ ID NO: 3500 actccagaactcaagtctt 1625 1644 SEQ ID NO: 4841 aagatcgcagactttgagt 11654 11673 1 3 SEQ ID NO: 3501 tccagaactcaagtcttca 1627 1646 SEQ ID NO: 4842 tgaactcagaagaattgga 1920 1939 1 3 SEQ ID NO: 3502 aagtacaaagccatcactg 1663 1682 SEQ ID NO: 4843 cagtcatgtagaaaaactt 1429 4448 1 3 SEQ ID NO: 3503 gccatcactgatgatccag 1672 1691 SEQ ID NO: 4844 ctggaactctctccatggc 10883 10902 1 3 SEQ ID NO: 3504 ccatcactgatgatccaga 1673 1692 SEQ ID NO: 4845 tctgaactcagaaggatgg 13999 14018 1 3 SEQ ID NO: 3505 atccagaaagctgccatcc 1685 1704 SEQ ID NO: 4846 ggatttcctaaagctggat 11173 11192 1 3 SEQ ID NO: 3506 cagaaagctgccatccagg 1699 1707 SEQ ID NO: 4847 cctgaaatacaatgctctg 5518 5537 1 3 SEQ ID NO: 3507 acaaggaccaggaggttct 1731 1750 SEQ ID NO: 4848 agaaacagcatttgtttgt 4542 4561 1 3 SEQ ID NO: 3508 aggaccaggaggttcttct 1734 1753 SEQ ID NO: 4849 agaagctaagcaatgtcct 7242 7261 1 SEQ ID NO: 3509 accaggaggttcttcttca 1737 1756 SEQ ID NO: 4850 tgaaggctgactctgtggt 4290 4309 1 3 SEQ ID NO: 3510 tcttcagactttccttgat 1750 1769 SEQ ID NO: 4851 atcaggaagggctcaaaga 2567 2586 1 3 SEQ ID NO: 3511 ttcagactttccttgatga 1752 1771 SEQ ID NO: 4852 tcattactcctgggctgaa 11307 11326 1 3 SEQ ID NO: 3512 gttgatgaggagtccttca 1810 1829 SEQ ID NO: 4853 tgaatctggctccctcaac 9046 9065 1 3 SEQ ID NO: 3513 cttcacaggcagatattaa 1824 1843 SEQ ID NO: 4854 ttaatcgagaggtatgaag 7148 7187 1 3 SEQ ID NO: 3514 ttcacaggcagatattaac 1825 1844 SEQ ID NO: 4855 gttaatcgagaggtatgaa 714 7 7166 1 3 SEQ ID NO: 3515 ggcagatattaacaaaatt 1831 1850 SEQ ID NO: 4856 aattgcattagatgatgcc 6589 6608 1 3 SEQ ID NO: 3516 atattaacaaaattgtcca 1836 1855 SEQ ID NO: 4857 tggagtttgtgacaaatat 2760 2779 1 3 SEQ ID NO: 3517 acaaaattgtccaaattct 1842 1861 SEQ ID NO: 4858 agaaacagcatttgtttgt 4542 4561 1 3 SEQ ID NO: 3518 gagcaagtgaagaactttg 1877 1896 SEQ ID NO: 4859 caaatgacatgatgggctc 5334 53 53 1 3 SEQ ID NO: 3519 gtgaagaactttgtggctt 1883 1902 SEQ ID NO: 4860 aagcatctgattgactcac 12677 12696 1 3 SEQ ID NO: 3520 agaactttgtggcttccca 1887 1906 SEQ ID NO: 4861 tgggcctgccccagattct 8909 8928 1 3 SEQ ID NO: 3521 tttgtggcttcccatattg 1892 1911 SEQ ID NO: 4862 caataagatcaatagcaaa 8998 9017 1 3 SEQ ID NO: 3522 tggcttcccatattgccaa 1896 1915 SEQ ID NO: 4863 ttggctcacatgaaggcca 7631 7650 1 3 SEQ ID NO: 3523 ttcccatattgccaatatc 1900 1919 SEQ ID NO: 4864 gatatacactagggaggaa 1274 5 12764 1 3 SEQ ID NO: 3524 tcccatattgccaatatct 1901 1920 SEQ ID NO: 4865 agatcaaagttaattggga 12276 12295 1 3 SEQ ID NO: 3525 ttgccaatatcttgaactc 1908 1927 SEQ ID NO: 4866 gagtcccagtgcccagcaa 9352 9371 1 3 SEQ ID NO: 3526 ttggatatccaagatctga 1934 1953 SEQ ID NO: 4867 tcagtataagtacaaccaa 9400 9419 1 3 SEQ ID NO: 3527 tccaagatctgaaaaagtt 1941 1960 SEQ ID NO: 4868 aacttccaactgtcatgga 1986 2005 1 3 SEQ ID NO: 3528 ctgaaaaagttagtgaaag 1949 1968 SEQ ID NO: 4869 ctttgaagtcagtcttcag 7915 7934 1 3 SEQ ID NO: 3529 agttagtgaaagaagttct 1956 1975 SEQ ID NO: 4870 agaatctcaacttccaact 1978 1997 1 3 SEQ ID NO: 3530 aatctcaacttccaactgt 1980 1999 SEQ ID NO: 487i acaggggtcctttatgatt 12350 12359 1 3 SEQ ID NO: 3531 gtcatggacttcagaaaat 1997 2016 SEQ ID NO: 4872 atttgaaagaataaatgac 7036 7055 1 3 SEQ ID NO: 3532 tcaactctacaaatctgtt 2029 2048 SEQ ID NO: 4873 aacacattgaggctattga 6978 6997 1 3 SEQ ID NO: 3533 aactctacaaatctgtttc 2031 2050 SEQ ID NO: 4874 gaaaaaggggattgaagtt 10284 10303 1 3 SEQ ID NO: 3534 aaatagaagggaatcttat 2079 2098 SEQ ID NO: 4875 ataagcaaactgtcaattc 5457 5476 1 3 SEQ ID NO: 3535 agaagggaatcttatattt 2083 2102 SEQ ID NO: 4876 aaatgcactgctgcgttct 4900 4919 1 3 SEQ ID NO: 3536 gaagggaatcttatatttg 2084 2103 SEQ ID NO: 4877 caaaaacattttcaacttc 5287 5308 1 3 SEQ ID NO: 3537 tgatccaaataactacctt 2101 2120 SEQ ID NO: 4878 aaggaagaaagaaaaatca 3461 3480 1 3 SEQ ID NO: 3538 tggatttgcttcagctgac 2158 2177 SEQ ID NO: 4879 gtcagcccagttccttcca 10932 10951 1 3 SEQ ID NO: 3539 tttgcttcagctgacctca 2162 2181 SEQ ID NO: 4880 tgaggaaactcagatcaaa 12265 12284 1 3 SEQ ID NO: 3540 cttggaaggaaaaggcttt 2191 2210 SEQ ID NO: 4881 aaagcattggtagagcaag 7850 7869 1 3 SEQ ID NO: 3541 tggaaggaaaaggctttga 2193 2212 SEQ ID NO: 4882 tcaagtctgtgggattcca 4086 105 1 3 SEQ ID NO: 3542 ggctttgagccaacattgg 2204 2223 SEQ ID NO: 4883 ccaagaggtatttaaagcc 12958 12977 1 3 SEQ ID NO: 3543 tgagccaacattggaagct 2209 2228 SEQ ID NO: 4884 agctttctgccactgctca 13521 13540 1 3 SEQ ID NO: 3544 gagccaacattggaagctc 2210 2229 SEQ ID NO: 4885 gagctttctgccactgctc 13520 13539 1 3 SEQ ID NO: 3545 aacattggaagctcttttt 2215 2234 SEQ ID NO: 4886 aaaagaaacagcatttgtt 4539 4558 1 3 SEQ ID NO: 3546 tggaagctctttttgggaa 2220 2239 SEQ ID NO: 4887 ttccggcacgtgggttcca 3785 3804 1 3 SEQ ID NO: 3547 ctctttttgggaagcaagg 2226 2245 SEQ ID NO: 4888 ccttactgactttgcagag 7798 7817 1 3 SEQ ID NO: 3548 tttttgggaagcaaggatt 2229 2248 SEQ ID NO: 4889 aatcattgaaaaattaaaa 6730 6749 1 3 SEQ ID NO: 3549 ttttcccagacagtgtcaa 2247 2266 SEQ ID NO: 4890 ttgatgaaatcattgaaaa 6723 6742 1 3 SEQ ID NO: 3550 ttggctataccaaagatga 2331 2350 SEQ ID NO: 4891 tcattgctcccggagccaa 2676 2695 1 3 SEQ ID NO: 3551 ataccaaagatgataaaca 2337 2356 SEQ ID NO: 4892 tgttgcttttgtaaagtat 6280 8299 1 3 SEQ ID NO: 3552 gagcaggatatggtaaatg 2357 2376 SEQ ID NO: 4893 catttcagccttcgggctc 4262 4281 1 3 SEQ ID NO: 3553 atggtaaatggaataatgc 2366 2385 SEQ ID NO: 4894 gcatgcctagtttctccat 9954 9973 1 3 SEQ ID NO: 3554 tggtaaatggaataatgct 2367 2386 SEQ ID NO: 4895 agcacagtacgaaaaacca 10809 10828 1 3 SEQ ID NO: 3555 taaatggaataatgctcag 2370 2389 SEQ ID NO: 4896 ctgaaagagatgaaattta 13067 13086 1 3 SEQ ID NO: 3556 tggaataatgctcagtgtt 2374 2393 SEQ ID NO: 4897 aacagatttgaggattcca 7981 8000 1 3 SEQ ID NO: 3557 tcagtgttgagaagctgat 2385 2404 SEQ ID NO: 4898 atcacaactcctccactga 9542 9561 1 3 SEQ ID NO: 3558 cagtgttgagaagctgatt 2386 2405 SEQ ID NO: 4899 aatcacaactcctccactg 9541 9560 1 3 SEQ ID NO: 3559 agtgttgagaagctgatta 2387 2406 SEQ ID NO: 4900 taatcacaactcctccact 9540 9559 1 3 SEQ ID NO: 3560 gattaaagatttgaaatcc 2401 2420 SEQ ID NO: 4901 ggatactaagtaccaaatc 6874 6893 1 3 SEQ ID NO: 3561 gatttgaaatccaaagaag 2408 2427 SEQ ID NO: 4902 cttccgtttaccagaaatc 8248 8267 1 3 SEQ ID NO: 3562 atttgaaatccaaagaagt 2409 2428 SEQ ID NO: 4903 acttccgtttaccagaaat 8247 8266 1 3 SEQ ID NO: 3563 atccaaagaagtcccggaa 2416 2435 SEQ ID NO: 4904 ttccaatttccctgtggat 3688 3707 1 3 SEQ ID NO: 3564 tccaaagaagtcccggaag 247 2436 SEQ ID NO: 4905 cttccaatttccctgtgga 3687 3706 1 3 SEQ ID NO: 3565 agagcctacctccgcatct 2438 2457 SEQ ID NO: 4906 agattaatccgctggctct 8571 8590 1 3 SEQ ID NO: 3566 gagcctacctccgcatctt 2439 2458 SEQ ID NO: 4907 aagattaatccgctggctc 8570 8589 1 3 SEQ ID NO: 3567 cttgggagaggagcttggt 2455 2474 SEQ ID NO: 4908 accactgggacctaccaag 1252 7 12546 1 3 SEQ ID NO: 3568 ggagcttggttttgccagt 2464 2483 SEQ ID NO: 4909 actggtggccaaaccctcc 2734 2753 1 3 SEQ ID NO: 3569 ttggttttgccagtctcca 2469 2488 SEQ ID NO: 4910 tggagaagccacactccaa 10771 10790 1 3 SEQ ID NO: 3570 cagtctccatgacctccag 2479 2498 SEQ ID NO: 4911 ctggtcgcctgccaaactg 3538 3557 1 3 SEQ ID NO: 3571 ctccatgacctccagctcc 2483 2502 SEQ ID NO: 4912 ggagtcattgctcccggag 2672 2691 1 3 SEQ ID NO: 3572 ctgggaaagctgcttctga 2501 2520 SEQ ID NO: 4913 tcagaaagctaccttccag 7939 7958 1 3 SEQ ID NO: 3573 gaggtcatcaggaagggct 2561 2580 SEQ ID NO: 4914 agccagaagtgagatcctc 3514 3533 1 3 SEQ ID NO: 3574 aagaatgacttttttcttc 2582 2601 SEQ ID NO: 4915 gaaggcatctgggagtctt 3835 3854 1 3 SEQ ID NO: 3575 cttttttcttcactacatc 2590 2609 SEQ ID NO: 4916 gatgcttacaacactaaag 6107 6126 1 3 SEQ ID NO: 3576 catcttcatggagaatgcc 2605 2624 SEQ ID NO: 4917 ggcacttccaaaattgatg 10718 10737 1 3 SEQ ID NO: 3577 cttcctggagaatgccttt 2608 2627 SEQ ID NO: 4918 aaagttaattgggaagaag 12281 12300 1 3 SEQ ID NO: 3578 aatgcctttgaactcccca 2618 2637 SEQ ID NO: 4919 tgggctggcttcagccatt 5737 5756 1 3 SEQ ID NO: 3579 gcctttgaactccccactg 2621 2640 SEQ ID NO: 4920 cagtctgaacattgcaggc 5383 5402 1 3 SEQ ID NO: 3580 caaggctggagtaaaactg 2692 2711 SEQ ID NO: 4921 cagtgcaacgaccaacttg 5080 5099 1 3 SEQ ID NO: 3581 tggagtaaaactggaagta 2698 2717 SEQ ID NO: 4922 tactcccacgccagctcca 3059 3078 1 3 SEQ ID NO: 3582 ggaagtagccaacatgcag 2710 2729 SEQ ID NO: 4923 ctgccatctcgagagttcc 4106 4125 1 3 SEQ ID NO: 3583 tttgtgacaaatatgggca 2765 2784 SEQ ID NO: 4924 tgcctttgtgtacaccaaa 11236 11255 1 3 SEQ ID NO: 3584 tgtgacaaatatgggcatc 2767 2786 SEQ ID NO: 4925 gatgggtctctacgccaca 4385 4404 1 3 SEQ ID NO: 3585 ggacttcgctaggagtggg 2794 2813 SEQ ID NO: 4926 cccaaggccacaggggtcc 12341 12360 1 3 SEQ ID NO: 3586 gtggggtccagatgaacac 2808 2827 SEQ ID NO: 4927 gtgttctagacctctccac 4179 1 198 1 3 SEQ ID NO: 3587 ttccacgagtcgggtctgg 2834 2853 SEQ ID NO: 4928 ccagaatctgtaccaggaa 12562 12581 1 3 SEQ ID NO: 3588 agtcgggtctggaggctca 2841 2860 SEQ ID NO: 4929 tgagaactacgagctgact 4807 4826 1 3 SEQ ID NO: 3589 tcgggtctggaggctcatg 2843 2862 SEQ ID NO: 4930 catgaaggccaaattccga 7639 7658 1 3 SEQ ID NO: 3590 aaaagctgggaagctgaag 2869 2888 SEQ ID NO: 4931 cttccagacacctgatttt 7951 7970 1 3 SEQ ID NO: 3591 aagctgaagtttatcattc 2879 2898 SEQ ID NO: 4932 gaatttacaattgttgctt 6269 6288 1 3 SEQ ID NO: 3592 gagaccagtcaagctgctc 2908 2927 SEQ ID NO: 4933 gagctccaggaagcttctc 13214 13233 1 3 SEQ ID NO: 3593 gcaacacattacatttggt 2934 2953 SEQ ID NO: 4934 accagtcagatattgttgc 10191 10210 1 3 SEQ ID NO: 3594 acattacatttggtctcta 2939 2958 SEQ ID NO: 4935 tagaatatgaactaaatgt 11889 11908 1 3 SEQ ID NO: 3595 cattacatttggtctctac 2940 2959 SEQ ID NO: 4936 gtagctgagaaaatcaatg 71 06 712 5 1 3 SEQ ID NO: 3596 aaacggaggtgatcccacc 2964 2983 SEQ ID NO: 4937 ggtggataccctgaagttt 3205 3224 1 3 SEQ ID NO: 3597 attgagaacaggcagtcct 2987 3006 SEQ ID NO: 4938 aggaaaagcgcacctcaat 12031 12050 1 3 SEQ ID NO: 3598 tgagaacaggcagtcctgg 2989 3008 SEQ ID NO: 4939 ccagcttccccacatctca 8341 8360 1 3 SEQ ID NO: 3599 ctgcacctcaggcgcttac 3043 3062 SEQ ID NO: 4940 gtaagaaaatacagagcag 6440 6459 1 3 SEQ ID NO: 3600 tccacagactccgcctcct 3074 3093 SEQ ID NO: 4941 aggacagagccttggtgga 3192 3211 1 3 SEQ ID NO: 3601 ctgaccggggacaccagat 3101 3120 SEQ ID NO: 4942 atctgatgaggaaactcag 12259 12278 1 3 SEQ ID NO: 3602 tagagctggaactgaggcc 3120 3139 SEQ ID NO: 4943 ggcctctctggggcatcta 5144 5163 1 3 SEQ ID NO: 3603 ctatgagctccagagagag 3175 3194 SEQ ID NO: 4944 ctctcacaaaaaagtatag 6549 6568 1 3 SEQ ID NO: 3604 cttggtggataccctgaag 3202 3221 SEQ ID NO: 4945 cttcaggaagcttctcaag 13217 13236 1 3 SEQ ID NO: 3605 ttgtaactcaagcagacgg 3222 3241 SEQ ID NO: 4946 ccttacacaataatcacaa 9530 9549 1 3 SEQ ID NO: 3606 1aactcaagcagaaggtgc 3225 3244 SEQ ID NO: 4947 gcacctagctggaaagtta 6955 6974 1 3 SEQ ID NO: 3607 gcagaaggtgcgaagcaga 3233 3252 SEQ ID NO: 4948 tctgtgggattccatctgc 4091 4 110 1 3 SEQ ID NO: 3608 cagaaggtgcgaagcagac 3234 3253 SEQ ID NO: 4949 gtctgtgggattccatctg 4090 4109 1 3 SEQ ID NO: 3609 gtatgaccttgtccagtga 3288 3307 SEQ ID NO: 4950 tccccaacggagaacatac 10851 10870 1 3 SEQ ID NO: 3610 tatgaccttgtccagtgaa 3289 3308 SEQ ID NO: 4951 Ctcaccaacggagaacata 10850 10869 1 3 SEQ ID NO: 3611 gaagtccaaattccggatt 3305 3324 SEQ ID NO: 4952 aatctcaagctttctcttc 10052 10071 1 3 SEQ ID NO: 3612 gagggcaaaacgtcttaca 3371 3390 SEQ ID NO: 4953 Cgtacaactggtccgcctc 4215 4234 1 3 SEQ ID NO: 3613 agggcaaaacgtcttacag 3372 3391 SEQ ID NO: 4954 ctgttaggacaccagccct 4062 4081 1 3 SEQ ID NO: 3814 gactcaccctggacattca 3390 3409 SEQ ID NO: 4955 tgaaattcaatcacaagtc 9076 9095 1 3 SEQ ID NO: 3615 ctggacattcagaacaaga 3398 3417 SEQ ID NO: 4956 tcttttcttttcagcccag 9226 9245 1 3 SEQ ID NO: 3616 tcatgggcgacctaagttg 3435 3454 SEQ ID NO: 4957 caactgcagacatatatga 6635 6854 1 3 SEQ ID NO: 3617 tgggcgacctaagttgtga 3438 3457 SEQ ID NO: 4958 tcactccattaacctccca 6316 6335 1 3 SEQ ID NO: 3618 agttgtgacacaaaggaag 3449 3468 SEQ ID NO: 4959 cttcttttccaattgaact 13838 13857 1 3 SEQ ID NO: 3619 tgacacaaaggaagaaaga 3454 3473 SEQ ID NO: 4960 tcttcatcttcatctgtca 10220 10239 1 3 SEQ ID NO: 3620 gacacaaaggaagaaagaa 3455 3474 SEQ ID NO: 4961 ttcttcatcttcacctgtc 10219 10238 1 3 SEQ ID NO: 3621 ggaagaaagaaaaatcaag 3463 3482 SEQ ID NO: 4962 cttgtcatgcctacgttcc 11348 11367 1 3 SEQ ID NO: 3622 aaaatcaagggtgttattt 3473 3492 SEQ ID NO: 4963 aaatcttattggggatttt 7084 7103 1 3 SEQ ID NO: 3623 tccataccccgtttgcaag 3491 3510 SEQ ID NO: 4964 cttggattcaaaatgtgga 6858 6877 1 3 SEQ ID NO: 3624 tgcaagcagaagccagaag 3504 3523 SEQ ID NO: 4965 cttcagggaacacaatgca 5185 5204 1 3 SEQ ID NO: 3625 cagaagccagaagtgagat 3510 3529 SEQ ID NO: 4966 atctatgccatctcttctg 5633 5652 1 3 SEQ ID NO: 3626 tgagatcctcgcccactgg 3523 3542 SEQ ID NO: 4967 ccagcttccccacatctca 8341 8360 1 3 SEQ ID NO: 3627 ggtcgcctgccaaactgct 3540 3559 SEQ ID NO: 4968 agcacatatgaactggacc 13947 13966 1 3 SEQ ID NO: 3628 tgcttctccaaatggactc 3555 3574 SEQ ID NO: 4969 gagtttatcagtcagagca 9701 9720 1 3 SEQ ID NO: 3629 tggactcatctgctacagc 3567 3586 SEQ ID NO: 4970 gctgcagtggcccgttcca 8167 8186 1 3 SEQ ID NO: 3630 gctacagcttatggctcca 3578 3597 SEQ ID NO: 4971 tggaggacattcctctagc 8211 8230 1 3 SEQ ID NO: 3631 ggtggcatggcattatgat 3610 3629 SEQ ID NO: 4972 atcacaaattagtttcacc 8947 8966 1 3 SEQ ID NO: 3632 agagaagattgaatttgaa 3631 3650 SEQ ID NO: 4973 ttcaacgatacctgtctct 7713 7732 1 3 SEQ ID NO: 3633 caggcaccaatgtagatac 3657 3676 SEQ ID NO: 4974 gtatgctaatagactcctg 3736 3755 1 3 SEQ ID NO: 3634 gacttccaatttccctgtg 3685 3704 SEQ ID NO: 4975 cacaatgcaaaattcagtc 5195 5214 1 3 SEQ ID NO: 3635 gtccctcaaacagacatga 3764 3783 SEQ ID NO: 4976 tcataagggaggtagggac 12777 12795 1 3 SEQ ID NO: 3636 caaacagacatgactttcc 3770 3789 SEQ ID NO: 4977 ggaactacaatttcatttg 7022 7041 1 3 SEQ ID NO: 3637 atagttgcaatgagctcat 3809 3828 SEQ ID NO: 4978 atgatttgaaaatagctat 6693 6 712 1 3 SEQ ID NO: 3638 gcttcagaaggcatctggg 3829 3848 SEQ ID NO: 4979 cccaagaggtatttaaagc 12957 12976 1 3 SEQ ID NO: 3639 ggagttcaacctccagaac 3895 3914 SEQ ID NO: 4980 gttcactccattaacctcc 6314 6333 1 3 SEQ ID NO: 3640 agaaaacctcttcttaaaa 3940 3959 SEQ ID NO: 4981 ttttctaaatggaacttct 12173 12192 1 3 SEQ ID NO: 3641 aaaacctcttcttaaaaag 3942 3961 SEQ ID NO: 4982 ctttgaaaaattctctttt 9213 9232 1 3 SEQ ID NO: 3642 aaaaagcgatggccgggtc 3955 3974 SEQ ID NO: 4983 gaccttgcaagaatatttt 6343 6362 1 3 SEQ ID NO: 3643 gtcaaatataccttgaaca 3971 3990 SEQ ID NO: 4984 tgttaacaaattccttgac 7363 73 82 1 3 SEQ ID NO: 3644 tgaacaagaacagtttgaa 3984 4003 SEQ ID NO: 4985 ttcaagttcctgaccttca 8310 8329 1 3 SEQ ID NO: 3645 agtttgaaaattgagattc 3995 4014 SEQ ID NO: 4986 gaatctggctccctcaact 9047 9066 1 3 SEQ ID NO: 3646 gtttgaaaattgagattcc 3996 4015 SEQ ID NO: 4987 ggaaataccaagtcaaaac 10454 104 73 1 3 SEQ ID NO: 3647 ttgaaaattgagattcctt 3998 4017 SEQ ID NO: 4988 aaggaaaagcgcacctcaa 12030 12049 1 3 SEQ ID NO: 3548 ctaaagatgttagagactg 4046 4065 SEQ ID NO: 4989 cagttgaccacaagcttag 10545 10564 1 3 SEQ ID NO: 3649 atgttagagactgttagga 4052 4071 SEQ ID NO: 4990 tccttaacaccttccacat 8073 8092 1 3 SEQ ID NO: 3650 cagccctccacttcaagtc 4074 4093 SEQ ID NO: 4991 gacttctctagtcaggctg 8813 8832 1 3 SEQ ID NO: 3651 agccctccacttcaagtct 4075 4094 SEQ ID NO: 4992 agacatcgctgggctggct 5728 5747 1 3 SEQ ID NO: 3652 ccatctgccatctcgagag 4102 4121 SEQ ID NO: 4993 ctctcaaatgacatgatgg 5330 5349 1 3 SEQ ID NO: 3653 attcccaagttgtatcaac 4142 4161 SEQ ID NO: 4994 gttgagaagccccaagaat 6254 6273 1 3 SEQ ID NO: 3654 tcaactgcaagtgcctctc 4156 4175 SEQ ID NO: 4995 gagatcaagacactgttga 8843 8862 1 3 SEQ ID NO: 3655 ggtgttctagacctctcca 4178 4197 SEQ ID NO: 4996 tggaaccctctccctcacc 4735 4754 1 3 SEQ ID NO: 3656 ctccacgaatgtctacagc 4192 4211 SEQ ID NO: 4997 gctggtaacctaaaaggag 5588 5607 1 3 SEQ ID NO: 3657 cacgaatgtctacagcaac 4195 4214 SEQ ID NO: 4998 gttgcccaccatcatcgtg 11671 11690 1 3 SEQ ID NO: 3658 acgaatgtctacagcaact 4196 4215 SEQ ID NO: 4999 agttgcccaccatcatcgt 11670 11689 1 3 SEQ ID NO: 3659 tcctccagtggtggcaaca 4232 4251 SEQ ID NO: 5000 tgttagttgctcttaagga 13359 13378 1 3 SEQ ID NO: 3660 cgttaccacatgaaggctg 4280 4299 SEQ ID NO: 5001 cagcaagtacctgagaacg 8611 8630 1 3 SEQ ID NO: 3661 gaaggctgactctgtggtt 4291 4310 SEQ ID NO: 5002 aacctatgccttaatcttc 13169 13188 1 3 SEQ ID NO: 3662 tgtggttgacctgctttcc 4303 4322 SEQ ID NO: 5003 ggaaagttaaaacaacaca 6965 6984 1 3 SEQ ID NO: 3663 cctgctttcctacaatgtg 4312 4331 SEQ ID NO: 5004 cacaccttgacattgcagg 11088 11107 1 3 SEQ ID NO: 3664 ctgctttcctacaatgtgc 4313 4332 SEQ ID NO: 5005 gcacaccttgacattgcag 11087 11106 1 3 SEQ ID NO: 3665 tcctacaatgtgcaaggat 4319 4338 SEQ ID NO: 5006 atccgctggctctgaagga 8577 8596 1 3 SEQ ID NO: 3666 tatgaccacaagaatacgt 4352 4371 SEQ ID NO: 5007 acgtccgtgtgccttcata 9984 10003 1 3 SEQ ID NO: 3567 atgaccacaagaatacgtc 4353 4372 SEQ ID NO: 5008 gacgtccgtgtgccttcat 9983 10002 1 3 SEQ ID NO: 3868 gaatacgtccacaccatca 4363 4382 SEQ ID NO: 5009 tgattatctgaattcattc 6487 6506 1 SEQ ID NO: 3669 tttctagattcgaatatca 4406 4425 SEQ ID NO: 5010 tgatttacatgatttgaaa 6685 6704 1 3 SEQ ID NO: 3670 gattcgaatatcaaattca 4412 4431 SEQ ID NO: 5011 tgaagtagctgagaaaatc 7102 7121 1 3 SEQ ID NO: 3671 gaaacaacccagtctcaaa 4449 4468 SEQ ID NO: 5012 tttgaaaaattctcttttc 9214 9233 1 3 SEQ ID NO: 3672 cccagtctcaaaaggttta 4456 4475 SEQ ID NO: 5013 taaattcattactcctggg 11302 11321 1 3 SEQ ID NO: 3673 ctcaaaaggtttactaata 4462 4481 SEQ ID NO: 5014 tattcaaaactgagttgag 12231 12250 1 3 SEQ ID NO: 3674 tcaaaaggtttactaatat 4463 4482 SEQ ID NO: 5015 atattcaaaactgagttga 12230 12249 1 3 SEQ ID NO: 3675 aaaaggtttactaatattc 4465 4484 SEQ ID NO: 5016 gaatttgaaagttcgtttt 9280 9299 1 3 SEQ ID NO: 3676 gaaacagcatttgtttgtc 4543 4562 SEQ ID NO: 5017 gacagcatcttcgtgtttc 11214 11233 1 3 SEQ ID NO: 3677 atttgtttgtcaaagaagt 4551 4570 SEQ ID NO: 5018 acttaaaaaatataaaaat 8022 8041 1 3 SEQ ID NO: 3678 tcaagattgatgggcagtt 4569 4588 SEQ ID NO: 5019 aactctcaagtcaagttga 13422 13441 1 3 SEQ ID NO: 3679 ttcagagtctcttcgttct 4586 4605 SEQ ID NO: 5020 agaagatggcaaatttgaa 11995 12014 1 3 SEQ ID NO: 3680 cagagtctcttcgttctat 4588 4607 SEQ ID NO: 5021 atagcatggacttcttctg 8873 8892 1 3 SEQ ID NO: 3681 atgctaaaggcacatatgg 4605 4624 SEQ ID NO: 5022 ccatttgagatcacggcat 9245 9264 1 3 SEQ ID NO: 3682 gcacatatggcctgtcttg 4614 4633 SEQ ID NO: 5023 caagttggcaagtaagtgc 9372 9391 1 3 SEQ ID NO: 3683 gagtccaacctgaggttta 4667 4686 SEQ ID NO: 5024 taaagtgccacttttactc s:90 6209 1 3 SEQ ID NO: 3684 agtccaacctgaggtttaa 4668 4687 SEQ ID NO: 5025 ttaacagggaagatagact 9308 9327 1 3 SEQ ID NO: 3685 cctacctccaaggcaccaa 4692 4711 SEQ ID NO: 5026 ttggcaagtaagtgctagg 9376 9395 1 3 SEQ ID NO: 3686 gaagatggaaccctctccc 4730 4749 SEQ ID NO: 5027 gggaagaagaggcagcttc 12291 12310 1 3 SEQ ID NO: 3687 tgatctgcaaagtggcatc 4762 4781 SEQ ID NO: 5028 gatgaggaaactcagatca 12263 12282 1 3 SEQ ID NO: 3688 gatctgcaaagtggcatca 4763 4782 SEQ ID NO: 5029 tgatgaggaaactcagatc 12262 12281 1 3 SEQ ID NO: 3689 gcttccctaaagtatgaga 4793 4812 SEQ ID NO: 5030 tctcgtgtctaggaaaagc 5977 5996 1 3 SEQ ID NO: 3690 gtatgagaactacgagctg 4804 4823 SEQ ID NO: 5031 cagcttaagagacacatac 6920 6939 1 3 SEQ ID NO: 3691 tctaacaagatggatatga 4868 4887 SEQ ID NO: 5032 tcattttccaactaataga 13032 13051 1 3 SEQ ID NO: 3692 ccgctgcgtcctgaatatc 4907 4926 SEQ ID NO: 5033 gatacaagaaaaactgcag 6901 6920 1 3 SEQ ID NO: 3693 tcattgaggttcttcagcc 4940 4959 SEQ ID NO: 5034 ggctcatatgctgaaatga 534 8 5367 1 3 SEQ ID NO: 3694 ttctggatcactaaattcc 4963 4982 SEQ ID NO: 5035 ggaaggacaaggcccagaa 12549 12568 1 3 SEQ ID NO: 3695 ccatggtcttgagttaaat 4981 5000 SEQ ID NO: 5036 atttttattcctgccatgg 10103 10122 1 3 SEQ ID NO: 3696 tcttaggcactgacaaaat 5007 5026 SEQ ID NO: 5037 attttttgcaagttaaaga 14019 14038 1 3 SEQ ID NO: 3697 acaaggcgacactaaggat 5040 5059 SEQ ID NO: 5038 atccatgatctacatttgt 6794 6813 1 3 SEQ ID NO: 3698 tgcaacgaccaacttgaag 5083 5102 SEQ ID NO: 5039 cttcagggaacacaatgca 5185 5204 1 3 SEQ ID NO: 3699 caacttgaagtgtagtctc 5092 5111 SEQ ID NO: 5040 gagatgagagatgccgttg 6239 6258 1 3 SEQ ID NO: 3700 gctggagaatgagctgaat 5116 5135 SEQ ID NO: 5041 attctcctttcttctcagc 9222 9241 1 3 SEQ ID NO: 3701 gcagagcttggcctctctg 5135 5154 SEQ ID NO: 5042 cagatacaagaaaaactgc 6899 6918 1 3 SEQ ID NO: 3702 tctctggggcatctatgaa 5148 5167 SEQ ID NO: 5043 11cattcaattgggagaga 6499 6518 1 3 SEQ ID NO: 3703 tccggggcacctatgaaat 5150 5169 SEQ ID NO: 5044 atttgtaagaaaatacaga 6436 6455 1 3 SEQ ID NO: 3704 aacacaatgcaaaattcag 5193 5212 SEQ ID NO: 5045 ctgaagcattaaaactgtt 7506 7525 1 3 SEQ ID NO: 3705 ctcacagagctatcactgg 5231 5250 SEQ ID NO: 5046 ccagatgctgaacagtgag 8149 8168 1 3 SEQ ID NO: 3706 tgggaagtgcttatcaggc 5247 5266 SEQ ID NO: 5047 gcctacgttccatgtccca 11356 11375 1 3 SEQ ID NO: 3707 ttcaaggtcagtcaagaag 5303 5322 SEQ ID NO: 5048 cttcagtgcagaatatgaa 11977 11996 1 3 SEQ ID NO: 3708 aatgacatgatgggctcat 5336 5355 SEQ ID NO: 5049 atgattatctgaattcatt 6486 6505 1 3 SEQ ID NO: 3709 gctcatatgctgaaatgaa 5349 5368 SEQ ID NO: 5050 ttcagccattgacatgagc 5746 5765 1 3 SEQ ID NO: 3710 acatgctgaaatgaaacct 5353 5372 SEQ ID NO: 5051 aaatagctattgctaatat 6702 6721 1 3 SEQ ID NO: 3711 tctgaacattgcaggctta 5386 5405 SEQ ID NO: 5052 taagaaccagaagatcaga 10996 11015 1 3 SEQ ID NO: 3712 gaacattgcaggcttatca 5389 5408 SEQ ID NO: 5053 tgatatcgacgtgaggttc 12490 12509 1 3 SEQ ID NO: 3713 tgcaggcttatcactggac 5395 5414 SEQ ID NO: 5054 gtcctggattccacatgca 11852 11871 1 3 SEQ ID NO: 3714 tcaaaacttgacaacattt 5420 5439 SEQ ID NO: 5055 aaattccttgacatgttga 7370 7389 1 3 SEQ ID NO: 3715 atttacagctctgacaagt 5435 5454 SEQ ID NO: 5056 acttaaaaaatataaaaat 8022 8041 1 3 SEQ ID NO: 3716 ctctgacaagttttataag 5443 5462 SEQ ID NO: 5057 cttacttgaattccaagag 10674 10693 1 3 SEQ ID NO: 3717 gttaatttacagctacagc 5468 5487 SEQ ID NO: 5058 gctgcatgtggctggtaac 5578 5597 1 3 SEQ ID NO: 3718 ttctctggtaactacttta 5491 5510 SEQ ID NO: 5059 taaaagattactttgagaa 7275 72 94 1 3 SEQ ID NO: 3719 cctaaaaggagcctaccaa 5596 5615 SEQ ID NO: 5060 ttggcaagtaagtgctagg 9376 9395 1 3 SEQ ID NO: 3720 aaaaggagcctaccaaaat 5599 5618 SEQ ID NO: 5061 atttacaattgttgctttc 6271 6290 1 3 SEQ ID NO: 3721 aggagcctaccaaaataat 5602 5621 SEQ ID NO: 5062 attacctatgatttctcct 10127 10146 1 3 SEQ ID NO: 3722 ataatgaaataaaacacat 5616 5635 SEQ ID NO: 5063 atgtcaaacactttgttat 7065 7084 1 3 SEQ ID NO: 3723 aaaacacatctatgccatc 5626 5645 SEQ ID NO: 5064 gatgaagatgacgactttt 12158 12177 1 3 SEQ ID NO: 3724 tgctaaggttcagggtgtg 5686 5705 SEQ ID NO: 5085 cacaagtcgattcccagca 9087 9106 1 3 SEQ ID NO: 3725 gagtttagccatcggctca 5705 5724 SEQ ID NO: 5066 tgaggtgactcagagactc 7450 7469 1 3 SEQ ID NO: 3726 gctggcttcagccattgac 5740 5759 SEQ ID NO: 5067 gtcagtgaagttctccagc 8596 8615 1 3 SEQ ID NO: 3727 atttcagcaatgtcttccg 5790 5809 SEQ ID NO: S068 cggagcatgggagtgaaat 8628 8647 1 3 SEQ ID NO: 3728 tttcagcaatgtcttccgt 5791 5810 SEQ ID NO: 5069 acggagcatgggagtgaaa 8627 8646 1 3 SEQ ID NO: 3729 ttcagcaatgtcttccgct 5792 5811 SEQ ID NO: 5070 aacggagcatgggagtgaa 8626 8645 1 3 SEQ ID NO: 3730 cagcaatgtcctccgttct 5794 5813 SEQ ID NO: 507l agaagtgtcttcaaagctg 12412 12431 1 3 SEQ ID NO: 3731 tgtcttccgttctgtaatg 5800 5819 SEQ ID NO: 5072 cattcaattgggagagaca 6501 6520 1 3 SEQ ID NO: 3732 gtcttccgttctgtaatgg 5801 5820 SEQ ID NO: 5073 ccattcagtctctcaagac 12975 12994 1 3 SEQ ID NO: 3733 atgggaaactcgctctctg 5859 5878 SEQ ID NO: 5074 cagataaaaaactcaccat 12213 12232 1 3 SEQ ID NO: 3734 ggagaacatactgggcagc 5879 5898 SEQ ID NO: 5075 gctgttttgaagactctcc 1088 1107 1 3 SEQ ID NO: 3735 gttgaaagcagaacctctg 5914 5933 SEQ ID NO: 5076 cagaattcataatcccaac 8274 82 93 1 3 SEQ ID NO: 3736 gtctaggaaaagcatcagt 5983 6002 SEQ ID NO: 5077 actgcaagatttttcagac 13612 13631 1 3 SEQ ID NO: 3737 agcatcagtgcagctcttg 5993 6012 SEQ ID NO: 5078 caagaacctgttagttgct 13351 13370 1 3 SEQ ID NO: 3738 ttgaacacaaagtcagtgc 6009 6028 SEQ ID NO: 5079 gcacatcaatattgatcaa 6418 6437 1 3 SEQ ID NO: 3739 gcagacaggcacctggaaa 6046 6065 SEQ ID NO: 5080 tttcagatggcattgctgc 11610 11629 1 3 SEQ ID NO: 3740 gaaactcaagacccaattt 6061 6080 SEQ ID NO: 5081 aaatcccatccaggttttc 8037 8056 1 3 SEQ ID NO: 3741 acaatgaatacagccagga 6084 6103 SEQ ID NO: 5082 tcctttggctgtgctttgt 9682 9701 1 3 SEQ ID NO: 3742 cttggatgcttacaacact 6103 6122 SEQ ID NO: 5083 agtgaagttctccagcaag 8599 8618 1 3 SEQ ID NO: 3743 ttggcgtggagcttactgg 6132 6151 SEQ ID NO: 5084 ccagaattcataatcccaa 8273 82 92 1 3 SEQ ID NO: 3744 cacttttactcagtgagcc 6198 6217 SEQ ID NO: 5085 ggctattgatgttagagtg 6988 7007 1 3 SEQ ID NO: 3745 tttagagatgagagatgcc 6235 6254 SEQ ID NO: 5086 ggcatgatgctcatttaaa 9177 9196 1 3 SEQ ID NO: 3746 gagaagccccaagaattta 6257 6276 SEQ ID NO: 5087 taaagccattcagtctctc 12970 12989 1 3 SEQ ID NO: 3747 caattgttgcttttgtaaa 6276 6295 SEQ ID NO: 5088 Cttaaccagtcagatattg 10187 10206 1 3 SEQ ID NO: 3748 ttttgtaaagtatgataaa 6286 6305 SEQ ID NO: 5089 tttattgctgaatccaaaa 13655 13674 1 3 SEQ ID NO: 3749 ttgtaaagtatgataaaaa 6288 6307 SEQ ID NO: 5090 ttttgagaggaatcgacaa 6358 6377 1 3 SEQ ID NO: 3750 ttcactccattaacctccc 6315 6334 SEQ ID NO: 5091 gggaaaaaacaggcttgaa 9576 9595 1 3 SEQ ID NO: 3751 ttttgagaccttgcaagaa 6337 6356 SEQ ID NO: 5092 ttctctctatgggaaaaaa 9566 9585 1 3 SEQ ID NO: 3752 accttgcaagaatattttg 6344 6363 SEQ ID NO: 5093 caaaagaagcccaagaggt 12948 12967 1 3 SEQ ID NO: 3753 tcaatattgaccaatttgt 6423 6442 SEQ ID NO: 5094 acaaagcagattatgttga 11829 11848 1 3 SEQ ID NO: 3754 cagagcagccctgggaaaa 6451 6470 SEQ ID NO: 5095 ttttcagaccaactctctg 13622 13641 1 3 SEQ ID NO: 3755 cctgggaaaactcccacag 6460 6479 SEQ ID NO: 5096 ctgtctctggtcagccagg 7724 7743 1 3 SEQ ID NO: 3756 actcccacagcaagctaat 6469 6488 SEQ ID NO: 5097 attacacttcctttcgagt 12869 12888 1 3 SEQ ID NO: 3757 aattcattcaattgggaga 6497 6516 SEQ ID NO: 5098 tctcttcctccatggaatt 10479 10498 1 3 SEQ ID NO: 3758 ttcaattgggagagacaag 6503 6522 SEQ ID NO: 5099 cttggagtgccagtttgaa 11808 11827 1 3 SEQ ID NO: 3759 aggagaaactgactgctct 6534 6553 SEQ ID NO: 5100 agagcttatgggatttcct 11163 11182 1 3 SEQ ID NO: 3760 actgactgctctcacaaaa 6541 6560 SEQ ID NO: 5101 ttttggcaagctatacagt 8380 8399 1 3 SEQ ID NO: 3761 gactgctctcacaaaaaag 6544 6563 SEQ ID NO: 5102 ctttgtgagtttatcagtc 9695 9714 1 3 SEQ ID NO: 3762 cagacatatatgatacaat 6641 6660 SEQ ID NO: 5103 attggatatccaagatctg 1933 1952 1 3 SEQ ID NO: 3763 aatttgatcagtatattaa 6657 6676 SEQ ID NO: 5104 ttaaaagaaatcttcaatt 13815 13834 1 3 SEQ ID NO: 3764 tatgatttacatgatttga 6683 6702 SEQ ID NO: 5105 tcaatgattatatcccata 13128 13147 1 3 SEQ ID NO: 3765 tttgaaaatagctattgct 6697 6716 SEQ ID NO: 5106 agcacagaaaaaattcaaa 13864 13883 1 3 SEQ ID NO: 3766 ttgaaaatagctattgcta 6698 6717 SEQ ID NO: 5107 tagcacagaaaaaattcaa 13863 13882 1 3 SEQ ID NO: 3767 aatagctattgctaatatt 6703 6722 SEQ ID NO: 5108 aataaatggagtctttatt 14084 14103 1 3 SEQ ID NO: 3768 attattgatgaaatcattg 6719 6738 SEQ ID NO: 5109 caataccagaattcataat 8268 8287 1 3 SEQ ID NO: 3769 aaagtcttgatgagcacta 6747 6766 SEQ ID NO: 5110 tagtgattacacttccttt 12864 12883 1 3 SEQ ID NO: 3770 aagtcttgatgagcactat 6748 6767 SEQ ID NO: Slll atagcaacactaaatactc 8769 8788 1 3 SEQ ID NO: 3771 ttgatgagcactatcatat 6753 6772 SEQ ID NO: 5112 atatccaagatgagatcaa 13101 13120 1 3 SEQ ID NO: 3772 taattttagtaaaaacaat 6777 6796 SEQ ID NO: 5113 attgagattccctccatta 11702 11721 1 3 SEQ ID NO: 3773 ttttagtaaaaacaatcca 6780 6799 SEQ ID NO: 5114 tggagtgccagtttgaaaa 11810 11829 1 3 SEQ ID NO: 3774 acatttgtttattgaaaat 6805 6824 SEQ ID NO: 5115 atttcctaaagctggatgt 11175 11194 1 3 SEQ ID NO: 3775 attgattttaacaaaagtg 6824 6843 SEQ ID NO: 5116 cactgttccagttgtcaat 9871 9890 1 3 SEQ ID NO: 3776 actttaacaaaagtggaag 6828 6847 SEQ ID NO: 5117 cttcaaagacttaaaaaat 8014 8033 1 3 SEQ ID NO: 3777 aaatcagaatccagataca 6888 6907 SEQ ID NO: 5118 tgtaccataagccatattt 10088 10107 1 3 SEQ ID NO: 3778 gaatccagatacaagaaaa 6894 6913 SEQ ID NO: 5119 ttttctaaacttgaaattc 9065 9084 1 3 SEQ ID NO: 3779 ttaagagacacatacagaa 6924 6943 SEQ ID NO: 5120 ttcttaaacattcctttaa 9491 9510 1 3 SEQ ID NO: 3780 atccagcacctagctggaa 6950 6969 SEQ ID NO: 5121 ttccaatttccctgtggat 3688 3707 1 3 SEQ ID NO: 3781 tgagcatgtcaaacacttt 7060 7079 SEQ ID NO: 5122 aaagtgccacttttactca 6191 6210 1 3 SEQ ID NO: 3782 gagcatgtcaaacactttg 7061 7080 SEQ ID NO: 5123 caaatgacatgatgggctc 5334 5353 1 3 SEQ ID NO: 3783 aaacactttgttataaatc 7070 7089 SEQ ID NO: 5124 gattatatcccatatgttt 13133 13152 1 3 SEQ ID NO: 3784 tgagaaaatcaatgccttc 7111 7130 SEQ ID NO: 5125 gaaggaaaagcgcacctca 12029 12048 1 3 SEQ ID NO: 3785 tatgaagtagaccaacaaa 7160 7179 SEQ ID NO: 5126 tttgtggagggtagtcata 10331 10350 1 3 SEQ ID NO: 3786 aagtagaccaacaaatcca 7164 7183 SEQ ID NO: 5127 tggatgaagatgacgactt 12156 12175 1 3 SEQ ID NO: 3787 aagttgaaggagactattc 7223 7242 SEQ ID NO: 5128 gaataccaatgctgaactt 10168 10187 1 3 SEQ ID NO: 3788 acaagttaagataaaagat 7264 7283 SEQ ID NO: 5129 atctaaattcagttcttgt 11334 11353 1 3 SEQ ID NO: 3789 aagataaaagattactttg 7271 7290 SEQ ID NO: 5130 caaaatagaagggaatctt 2077 2096 1 3 SEQ ID NO: 3790 gattactttgagaaattag 7290 7299 SEQ ID NO: 5131 ctaaacttgaaattcaatc 9069 9088 1 3 SEQ ID NO: 3791 tgagaaattagttggattt 7288 7307 SEQ ID NO: 5132 aaatccgtgaggtgactca 7443 7462 1 3 SEQ ID NO: 3792 aaattagttggatttattg 7292 7311 SEQ ID NO: 5133 caattttgagaatgaattt 10419 10438 1 3 SEQ ID NO: 3793 tggatttattgatgatgct 7300 7319 SEQ ID NO: 5134 agcatgcctagtttctcca 9953 9972 1 3 SEQ ID NO: 3794 tcattgaagatgttaacaa 7353 7372 SEQ ID NO: 5135 ttgtagatgaaaccaatga 7422 7441 1 3 SEQ ID NO: 3795 cattgaagatgttaacaaa 7354 7373 SEQ ID NO: 5136 Cttgtagatgaaaccaatg 7421 7440 1 3 SEQ ID NO: 3798 attgaagatgttaacaaat 7355 7374 SEQ ID NO: 5137 atttaagtatgatttcaat 10495 10514 1 3 SEQ ID NO: 3797 ttgaagatgttaacaaatt 7356 7375 SEQ ID NO: 5138 aatttaagtatgacttcaa 10494 10513 1 3 SEQ ID NO: 3798 tgaagatgttaacaaattc 7357 7376 SEQ ID NO: 5139 gaatttaagtatgatttca 10493 10512 1 3 SEQ ID NO: 3799 acatgttgataaagaaatt 7380 7399 SEQ ID NO: 5140 aattccctgaagttgatgt 11487 11508 1 3 SEQ ID NO: 3800 tttgattaccaccagtttg 7406 7425 SEQ ID NO: 5141 caaattgaacatccccaaa 8791 8810 1 3 SEQ ID NO: 3801 caaaatccgtgaggtgact 7441 7460 SEQ ID NO: 5142 agtccccctaacagatttg 7972 7991 1 3 SEQ ID NO: 3802 aaaatccgtgaggtgactc 7442 7461 SEQ ID NO: 5143 gagtgaaatgctgtttttt 8638 8657 1 3 SEQ ID NO: 3803 aggtgactcagagactcaa 7452 7471 SEQ ID NO: 5144 ttgatgatatctggaacct 10731 10750 1 3 SEQ ID NO: 3804 gtgaaattcaggctctgga 7473 7492 SEQ ID NO: 5145 tccaatctcctcttttcac 8409 8428 1 3 SEQ ID NO: 3805 gttgcagtgtatctggaaa 7547 7566 SEQ ID NO: 5146 tttcaagcaaatgcacaac 8540 8559 1 3 SEQ ID NO: 3806 ttaagttcagcatctttgg 7616 7635 SEQ ID NO: 5147 ccaatgctgaactttttaa 10173 10192 1 3 SEQ ID NO: 3807 tgaaggccaaattccgaga 7641 7660 SEQ ID NO: 5148 tctcccctcttcaccttca 10213 10232 1 3 SEQ ID NO: 3808 aatgtatcaaatggacatt 7684 7703 SEQ ID NO: 514  9aatgaagtccggattcatt 11021 11040 1 3 SEQ ID NO: 3809 attcagcaggaacttcaac 7700 7719 SEQ ID NO: 5150 gttgagaagccccaagaat 6254 6273 1 3 SEQ ID NO: 3810 acctgtctctggtcagcca 7722 7741 SEQ ID NO: 5151 tggcaagtaagtgctaggt 9377 9396 1 3 SEQ ID NO: 3811 cctgtctctggtcagccag 7723 7742 SEQ ID NO: 5152 ctggacttctctagtcagg 8810 8829 1 3 SEQ ID NO: 3812 ggtcagccaggtttatagc 7732 7751 SEQ ID NO: 5153 gctaaaggagcagttgacc 10535 10554 1 3 SEQ ID NO: 3813 ccaggtttatagcacactt 7738 7757 SEQ ID NO: 5154 aagtccggattcattctgg 11025 11044 1 3 SEQ ID NO: 3814 gtttatagcacacttgtca 7742 7761 SEQ ID NO: 5155 tgacctgtccattcaaaac 13681 13700 1 3 SEQ ID NO: 3815 acttgtcacctacatttct 7753 7772 SEQ ID NO: 5156 agaaaaaggggattgaagt 10283 10302 1 3 SEQ ID NO: 3816 ctgattggtggactcttgc 7770 7789 SEQ ID NO: 5157 gcaagttaaagaaaatcag 1402 6 14045 1 3 SEQ ID NO: 3817 atgaaagcattggtagagc 7847 7866 SEQ ID NO: 5158 gctcatctcctttcttcat 10208 10227 1 3 SEQ ID NO: 3818 tgaaagcattggtagagca 7848 7867 SEQ ID NO: 5159 tgctcatctcctttcttca 10207 10226 1 3 SEQ ID NO: 3819 gggttcactgttcctgaaa 7868 7887 SEQ ID NO: 5160 tttcaccatagaaggaccc 8959 8978 1 3 SEQ ID NO: 3820 tcaagaccatccttgggac 7887 7906 SEQ ID NO: 5161 gtccccctaacagatttga 7973 7992 1 3 SEQ ID NO: 3821 ccttgggaccatgcctgcc 7897 7916 SEQ ID NO: 5162 ggcaccagggctcggaagg 13978 13997 1 3 SEQ ID NO: 3822 ttcaggctcttcagaaagc 7929 7948 SEQ ID NO: 5163 gcttgaaggcattcttgaa 9588 9607 1 3 SEQ ID NO: 3823 ttcagataaacttcaaaga 8004 8023 SEQ ID NO: 5164 tcttcataagttcaatgaa 13183 13202 1 3 SEQ ID NO: 3824 acttcaaagacttaaaaaa 8013 8032 SEQ ID NO: 5165 ttttaacaaaagtggaagt 6829 6848 1 3 SEQ ID NO: 3825 atcccatccaggttttcca 8039 8058 SEQ ID NO: 5168 tggagaagcaaatctggat 9472 9491 1 3 SEQ ID NO: 3826 gaatttaccatccttaaca 8063 8082 SEQ ID NO: 5167 tgttgaagtgtctccattc 9889 9908 1 3 SEQ ID NO: 3827 cattccttcctttacaatt 8089 8108 SEQ ID NO: 5168 aattccaattttgagaatg 10414 10433 1 3 SEQ ID NO: 3828 ttgaccagatgctgaacag 8145 8164 SEQ ID NO: 5169 ctgttgaaagatttatcaa 12932 12951 1 3 SEQ ID NO: 3829 aatcaccctgccagacttc 8233 8252 SEQ ID NO: 5170 gaagttctcaattttgatt 8522 8541 1 3 SEQ ID NO: 3830 tgaccttcccataccagaa 8320 8339 SEQ ID NO: 517i ttcttctggaaaagggtca 8884 8903 1 3 SEQ ID NO: 3831 ttccagcttccccacatct 8339 8358 SEQ ID NO: 5172 agattctcagatgagggaa 8921 8940 1 3 SEQ ID NO: 3832 aagctatacagtattctga 8387 8406 SEQ ID NO: 5173 tcagatggcattgctgctt 11612 11631 1 3 SEQ ID NO: 3833 attctgaaaatccaatctc 8399 8418 SEQ ID NO: 5174 gagataaccgtgcctgaat 11552 11571 1 3 SEQ ID NO: 3834 tttcacattagatgcaaat 8422 8441 SEQ ID NO: 5175 attttgaaaaaaacagaaa 9738 9757 1 3 SEQ ID NO: 3835 caaatgctgacatagggaa 8436 8455 SEQ ID NO: 5176 ttccatcacaaatcctttg 9670 9689 1 3 SEQ ID NO: 3836 gagagtccaaattagaagt 8508 8527 SEQ ID NO: 5177 actttacttcccaactctc 13410 13429 1 3 SEQ ID NO: 3837 agagtccaaattagaagtt 8509 8528 SEQ ID NO: 5178 aactttacttcccaactct 13409 13428 1 3 SEQ ID NO: 3838 tctcaattttgattttcaa 8527 8546 SEQ ID NO: 5179 Ctgatccccttttctgaga 11537 11556 1 3 SEQ ID NO: 3839 caattttgattttcaagca 8530 8549 SEQ ID NO: 5180 tgctgaatccaaaagattg 13660 13679 1 3 SEQ ID NO: 3840 aatgcacaaccctcaaacc 8549 8568 SEQ ID NO: 5181 ggtttatcaaggggccatt 12460 12479 1 3 SEQ ID NO: 3841 agttctccagcaagtacct 8604 8623 SEQ ID NO: 5182 aggttccatcgtgcaaact 11388 11407 1 3 SEQ ID NO: 3842 agtacctgagaacggagca 8516 8635 SEQ ID NO: 5183 tgctccaggagaacttact 13780 13799 1 3 SEQ ID NO: 3843 tcaaacacagtggcaagtt 8678 8697 SEQ ID NO: 5184 aactctcaagtcaagttga 13422 13441 1 3 SEQ ID NO: 3844 acaatcagcttaccctgga 8751 8770 SEQ ID NO: 5185 tccattctgaatatattgt 13380 133 99 1 3 SEQ ID NO: 3845 ctggatagcaacactaaat 8765 8784 SEQ ID NO: 5186 attttctgaacttccccag 12702 12721 1 3 SEQ ID NO: 3846 ctgacctgcgcaacgagat 8829 8848 SEQ ID NO: 5187 atctgatgaggaaactcag 12259 12278 1 3 SEQ ID NO: 3847 agatgagggaacacatgaa 8929 8948 SEQ ID NO: 5188 ttcatgtccctagaaatct 10038 10057 1 3 SEQ ID NO: 3848 tcaacttttctaaacttga 9060 9079 SEQ ID NO: 5189 tcaaggataacgtgtttga 12618 12637 1 3 SEQ ID NO: 3849 ttctaaacttgaaattcaa 9067 9086 SEQ ID NO: 5190 ttgatgatgctgtcaagaa 7308 7327 1 3 SEQ ID NO: 3850 gaaattcaatcacaagteg 9077 9096 SEQ ID NO: 5191 cgacgaagaaaatcattta 13566 13585 1 3 SEQ ID NO: 3851 cactgtttggagaagggaa 9141 9160 SEQ ID NO: 5192 ttccagaaagcagccagtg 12506 12525 1 3 SEQ ID NO: 3852 actgtttggagaagggaag 9142 9161 SEQ ID NO: 5193 cttccccaaagagaccagt 2898 2917 1 3 SEQ ID NO: 3853 aattctcttttcttttcag 9221 9240 SEQ ID NO: 5194 ctgattactatgaaaaatt 13638 13657 1 3 SEQ ID NO: 3854 ttcttttcagcccagccat 9230 9249 SEQ ID NO: 5195 atggaaaagggaaagagaa 13494 13513 1 3 SEQ ID NO: 3855 tttgaaagttcgttttcca 9283 9302 SEQ ID NO: 5196 tggaagtgtcagtggcaaa 10380 10399 1 3 SEQ ID NO: 3856 cagggaagatagacttcct 9312 9331 SEQ ID NO: 5197 aggacctttcaaattcctg 9848 9867 1 3 SEQ ID NO: 3857 ataagtacaaccaaaattt 9405 9424 SEQ ID NO: 5198 aaatcaggatctgagttat 14038 14057 1 3 SEQ ID NO: 3858 acaacgagaacattatgga 9435 9454 SEQ ID NO: 5199 tccattctgaatatattgt 13380 13399 1 3 SEQ ID NO: 3859 aggaataaatggagaagca 9463 9482 SEQ ID NO: 5200 tgctggaattgtcattcct 11734 11753 1 3 SEQ ID NO: 3860 agcaaatctggatttctta 9478 9497 SEQ ID NO: 5201 taagttctctgtacctgct 11719 11738 1 3 SEQ ID NO: 3861 tcctttaacaattcctgaa 9502 9521 SEQ ID NO: 5202 ttcaaaacgagcttcagga 13206 13225 1 3 SEQ ID NO: 3862 tttaacaattcctgaaatg 9505 9524 SEQ ID NO: 5203 catttgatttaagtgtaaa 9621 9540 1 3 SEQ ID NO: 3863 acacaataatcacaactcc 9534 9553 SEQ ID NO: 5204 ggagacagcatcttcgtgt 11211 11230 1 3 SEQ ID NO: 3864 aagatttctctctatggga 9561 9580 SEQ ID NO: 5205 tcccagaaaacctcttctt 3936 3955 1 3 SEQ ID NO: 3865 gaaaaaacaggcttgaagg 9578 9597 SEQ ID NO: 5206 ccttttacaattcattttc 13021 13040 1 3 SEQ ID NO: 3866 ttgaaggaattcttgaaaa 9590 9609 SEQ ID NO: 5207 ttttgagaatgaatttcaa 10422 10441 1 3 SEQ ID NO: 3867 tgaaggaattcttgaaaac 9591 9610 SEQ ID NO: 5208 gttttggctgataaattca 11291 11310 1 3 SEQ ID NO: 3868 agctcagtataagaaaaac 9640 9659 SEQ ID NO: 5209 gtttgataagtacaaagct 9805 9824 1 3 SEQ ID NO: 3869 tcaaatcctttgacaggca 9720 9739 SEQ ID NO: 5210 tgcctgagcagaccattga 11688 11707 1 3 SEQ ID NO: 3870 atgaaacaaaaattaagtt 9789 9808 SEQ ID NO: 5211 aactttgcactatgttcat 12762 12781 1 3 SEQ ID NO: 3871 aattcctggatacactgtt 9859 9878 SEQ ID NO: 5212 aacacatgaatcacaaatt 8938 8957 1 3 SEQ ID NO: 3872 ttccagttgtcaatgttga 9876 9895 SEQ ID NO: 5213 tcaaaacgagcttcaggaa 13207 13226 1 3 SEQ ID NO: 3873 aagtgtctccattcaccat 9894 9913 SEQ ID NO: 5214 atgggaagtataagaactt 4842 4861 1 3 SEQ ID NO: 3874 gtcagcatgcctagtttct 9950 9969 SEQ ID NO: 5215 agaaaaggcacaccttgac 11080 11099 1 3 SEQ ID NO: 3875 ctgccatgggcaatattac 10113 10132 SEQ ID NO: 5216 gtaagaaaatacagagcag 6440 6459 1 3 SEQ ID NO: 3876 tgaataccaatgctgaact 10167 10186 SEQ ID NO: 5217 agttgaaggagactattca 7224 7243 1 3 SEQ ID NO: 3877 tattgttgctcatctcctt 10201 10220 SEQ ID NO: 5218 aaggaaacataaactaata 12889 12908 1 3 SEQ ID NO: 3878 tgttgctcatctcctttct 10204 10223 SEQ ID NO: 5219 agaagaaatctgcagaaca 12431 12450 1 3 SEQ ID NO: 3879 tctgtcattgatgcactgc 10232 10251 SEQ ID NO: 5220 gcagtagactataagcaga 13928 13947 1 3 SEQ ID NO: 3880 ccacagctctgtctctgag 10305 10324 SEQ ID NO: 5221 ctcagggatctgaaggtgg 8195 8214 1 3 SEQ ID NO: 388l atttgtggagggtagtcat 10330 10349 SEQ ID NO: 5222 atgaagtagaccaacaaat 7161 7180 1 3 SEQ ID NO: 3882 atatggaagtgtcagtggc 10377 10396 SEQ ID NO: 5223 gccacactccaacgcatat 10778 10797 1 3 SEQ ID NO: 3883 tggaaataccaagtcaaaa 10453 10472 SEQ ID NO: 5224 ttttacaattcattttcca 13023 13042 1 3 SEQ ID NO: 3684 aagtcaaaacctactgtct 10463 10482 SEQ ID NO: 5225 agacctagtgattacactt 12859 12878 1 3 SEQ ID NO: 3885 actgtctcttcctccatgg 10475 10494 SEQ ID NO: 5226 ccatgcaagtcagcccagt 10924 10943 1 3 SEQ ID NO: 3886 cttcctccatggaatttaa 10482 10501 SEQ ID NO: 5227 ttaatcgagaggtatgaag 7148 7167 1 3 SEQ ID NO: 3887 attcttcaatgctgtactc 10512 10531 SEQ ID NO: 5228 gagttgagggtccgggaat 12242 12261 1 3 SEQ ID NO: 3888 tcgaccacaagcttagcct 10548 10567 SEQ ID NO: 5231 aagcgcacctcaacatcaa 12036 12055 1 3 SEQ ID NO: 3889 cctcacctcttacttttcc 10573 10592 SEQ ID NO: 5232 ggaactattgctagtgagg 10649 10668 1 3 SEQ ID NO: 3890 agctgcagggcacttccaa 10710 10729 SEQ ID NO: 5233 ttgggaagaagaggcagct 12289 12308 1 3 SEQ ID NO: 3891 ttccaaaattgatgatatc 10723 10742 SEQ ID NO: 5234 gatatacactagggaggaa 12745 12764 1 3 SEQ ID NO: 3892 gagaacatacaagcaaagc 10860 10879 SEQ ID NO: 5235 gcttggttttgccagtctc 2467 2486 1 3 SEQ ID NO: 3893 atggcaaatgtcagctctt 10897 10916 SEQ ID NO: 5236 acgaggtatttaaagccat 12960 12979 1 3 SEQ ID NO: 3894 tggcaaatgtcagctcttg 10898 10917 SEQ ID NO: 5237 caagaggtatttaaagcca 12959 12978 1 3 SEQ ID NO: 3895 ttgttcaggtccatgcaag 10914 10933 SEQ ID NO: 5238 cttgggggaggaggaacaa 14066 14085 1 3 SEQ ID NO: 3896 tgttcaggtccatgcaagt 10915 10934 SEQ ID NO: 5239 acttgggggaggaggaaca 14065 14084 1 3 SEQ ID NO: 3897 agttccttccatgatttcc 10940 10959 SEQ ID NO: 5240 ggaatctgatgaggaaact 12256 12275 1 3 SEQ ID NO: 3898 tgctaacactaagaaccag 10987 11006 SEQ ID NO: 5241 ctggatgtaaccaccagca 11186 11205 1 3 SEQ ID NO: 3899 actaagaaccagaagatca 10994 11013 SEQ ID NO: 5242 tgatcaagaacctgttagt 13347 13366 1 3 SEQ ID NO: 3900 ctaagaaccagaagatcag 10995 11014 SEQ ID NO: 5243 ctgatcaagaacctgttag 13346 13365 1 3 SEQ ID NO: 3901 cagaagatcagatggaaaa 11003 11022 SEQ ID NO: 5244 ttttcagaccaactctctg 13622 13641 1 3 SEQ ID NO: 3902 aaaaatgaagtccggattc 11018 11037 SEQ ID NO: 5245 gaatttgaaagttcgtttt 9280 9299 1 3 SEQ ID NO: 3903 gattcattctgggtctttc 11032 11051 SEQ ID NO: 5246 gaaaacctatgccttaatc 13166 13185 1 3 SEQ ID NO: 3994 aagaaaaggcacaccttga 11079 11098 SEQ ID NO: 5247 tcaaaacctactgtctctt 10466 10485 1 3 SEQ ID NO: 3905 aaggacacctaaggttcct 11115 11134 SEQ ID NO: 5248 aggacaccaaaataacctt 7572 7591 1 3 SEQ ID NO: 3906 ccagcattggtaggagaca 11199 11218 SEQ ID NO: 5249 tgtcaacaagtaccactgg 12370 12389 1 3 SEQ ID NO: 3907 ctttgtgtacaccaaaaac 11239 11258 SEQ ID NO: 5250 gtttttaaattgttgaaag 13148 13167 1 3 SEQ ID NO: 3908 ccatccctgtaaaagtttt 11277 11296 SEQ ID NO: 5251 aaaagggtcatggaaatgg 8893 8912 1 3 SEQ ID NO: 3909 tgatctaaattcagttctt 11332 11351 SEQ ID NO: 5252 aagatagtcagtctgatca 13334 13353 1 3 SEQ ID NO: 3910 aagaagctgagaacttcat 11432 11451 SEQ ID NO: 5253 atgagatcaacacaatctt 13110 13129 1 3 SEQ ID NO: 3911 tttgccctcaacctaccaa 11453 11472 SEQ ID NO: 5254 ttggtacgagttactcaaa 12641 12660 1 3 SEQ ID NO: 3912 cttgattcccttttttgag 11536 11555 SEQ ID NO: 5255 ctcaattttgattttcaag 8528 8547 1 3 SEQ ID NO: 3913 ttcacgcttccaaaaagtg 11591 11610 SEQ ID NO: 5256 cactcattgattttctgaa 12693 12712 1 3 SEQ ID NO: 3914 tgtttcagatggcattgct 11608 11627 SEQ ID NO: 5257 agcagattatgttgaaaca 11833 11852 1 3 SEQ ID NO: 3915 aatgcagtagccaacaaga 11639 11658 SEQ ID NO: 5258 tcttttcagcccagccatt 9231 9250 1 3 SEQ ID NO: 3916 ctgagcagaccattgagat 11691 11710 SEQ ID NO: 5259 atctgatgaggaaactcag 12259 12278 1 3 SEQ ID NO: 3917 tgagcagaccattgagatt 11692 11711 SEQ ID NO: 5260 aatctgatgaggaaactca 12258 12277 1 3 SEQ ID NO: 3918 ttgagattccctccattaa 11703 11722 SEQ ID NO: 5261 ttaatcttcataagttcaa 13179 13198 1 3 SEQ ID NO: 3919 acttggagtgccagtttga 11807 11826 SEQ ID NO: 5262 tcaattgggagagacaagt 6504 6523 1 3 SEQ ID NO: 3920 caaatttgaaggacttcag 12004 12023 SEQ ID NO: 5263 ctgagaacttcatcatttg 11438 11457 1 3 SEQ ID NO: 392l agcccagcgttcaccgatc 12056 12075 SEQ ID NO: 5264 gatccaagtatagttggct 13286 13305 1 3 SEQ ID NO: 3922 cagcgttcaccgatctcca 12060 12079 SEQ ID NO: 5265 tggacccgcaccaaagccg 13960 13979 1 3 SEQ ID NO: 3923 ctccatctgcgctaccaga 12074 12093 SEQ ID NO: 5266 tctgatatacatcacggag 13711 13730 1 3 SEQ ID NO: 3924 atgaggaaactcagatcaa 12264 12283 SEQ ID NO: 5267 ttgagttgcccaccateat 11667 1 1686 1 3 SEQ ID NO: 3925 aggcagcttctggcttgct 12300 12319 SEQ ID NO: 5268 agcaagtctttcctggcct 3018 3037 1 3 SEQ ID NO: 3926 tgaaagacaacgtgcccaa 12327 12346 SEQ ID NO: 5269 ttgggagagacaagtttca 6608 6527 1 3 SEQ ID NO: 3927 tatgattatgtcaacaagt 12362 12381 SEQ ID NO: 5270 actttgcacactgttcata 12763 12782 1 3 SEQ ID NO: 3928 cattaggcaaattgatgat 12475 12494 SEQ ID NO: 527l atcaacacaatcttcaatg 13115 13134 1 3 SEQ ID NO: 3929 ttgactcaggaaggccaag 12564 12603 SEQ ID NO: 5272 cttggtacgagttactcaa 12640 12659 1 3 SEQ ID NO: 3930 gaaacctgggatatacact 12736 12755 SEQ ID NO: 5273 agtgattacacttcctttc 12865 12884 1 3 SEQ ID NO: 3931 tcctttcqagttaaggaaa 12877 12896 SEQ ID NO: 5274 tttctgccactgctcagga 13524 13543 1 3 SEQ ID NO: 3932 gccattcagtctctcaaga 12974 12993 SEQ ID NO: 5275 tcttccgttctgtaatggc 5802 5821 1 3 SEQ ID NO: 3933 gtgctacgtaatcttcagg 13001 13020 SEQ ID NO: 5276 cctgcaccaaagctggcac 13964 13983 1 3 SEQ ID NO: 3934 agctgaaagagatgaaatt 13065 13084 SEQ ID NO: 5277 aatttattcaaaacgagct 13200 13219 1 3 SEQ ID NO: 3935 aatttacttatcttattaa 13080 13099 SEQ ID NO: 5278 ttaaaagaaatcttcaatt 13815 13834 1 3 SEQ ID NO: 3936 ttttaaattgttgaaagaa 13150 13169 SEQ ID NO: 5279 ttctctctatgggaaaaaa 9566 9585 1 3 SEQ ID NO: 3937 taatcttcataagttcaat 13180 13199 SEQ ID NO: 5280 attgagattccctccatta 11702 11721 1 3 SEQ ID NO: 3938 atattttgatccaagtata 13279 13298 SEQ ID NO: 5281 tataagcagaagcacatat 13937 13956 1 3 SEQ ID NO: 3939 tgaaatattatgaacttga 13311 13330 SEQ ID NO: 5282 tcaaccttaatgattttca 8295 8314 1 3 SEQ ID NO: 3940 caatttctgcacagaaata 13442 13461 SEQ ID NO: 5283 tattcttcttttccaattg 13834 13853 1 3 SEQ ID NO: 394l agaagattgcagagctttc 13509 13528 SEQ ID NO: 5284 gaaatcttcaatttattct 13821 13840 1 3 SEQ ID NO: 3942 gaagaaaataatttctgat 13570 13589 SEQ ID NO: 5285 atcagttcagataaacttc 7999 8018 1 3 SEQ ID NO: 3943 ttgacctgtccattcaaaa 13680 13699 SEQ ID NO: 5286 ttttgagaatgaatttcaa 10422 10441 1 3 SEQ ID NO: 3944 tcaaaactaccacacattt 13693 13712 SEQ ID NO: 5287 aaattccttgacatgttga 7370 7389 1 3 SEQ ID NO: 3945 ttttttaaaagaaatcttc 13811 13830 SEQ ID NO: 5288 gaagtgtcagtggcaaaaa 10382 10401 1 3 SEQ ID NO: 3946 aggatctgcgttattttgc 14043 14062 SEQ ID NO: 5289 gcaagggttcactgttcct 7864 7883 1 3 SEQ ID NO: 3947 tttgctaaacttgggggag 14057 14076 SEQ ID NO: 5290 ctccccaggacctttcaaa 9842 9861 1 3

TABLE 10 Start End Start End Source Index Index Match Index Index # B SEQ ID NO: 5291 tccatcttcaggaagctgt 222 241  SEQ ID NO: 5369 acagactctttcagatgga 1340 1359 1 6 SEQ ID NO: 5292 ccatcttcaggaagctgtg 223 242  SEQ ID NO: 5370 cacagactctttcagatgg 1339 1358 1 6 SEQ ID NO: 5293 cctctggccatgccatggg 417 436  SEQ ID NO: 5371 cccattttgaggccagagg 1492 1511 1 6 SEQ ID NO: 5294 ctctggccatgccatgggc 418 437  SEQ ID NO: 5372 gcccattttgaggccagag 1491 1610 1 6 SEQ ID NO: 5295 ttgaatgtcattttgtggt 521 540  SEQ ID NO: 5373 accatacattatcattcaa 2945 2964 1 6 SEQ ID NO: 5296 tcagtaatgggggaccagc 1886 1905  SEQ ID NO: 5374 gctggtctcgaactcctga 2731 2750 1 6 SEQ ID NO: 5297 ttttactgtgcatacatgt 1956 1975  SEQ ID NO: 5375 acatctttgaaaagaaaaa 2983 3002 1 6 SEQ ID NO: 5298 tgaggtgccaaggaaatga 50 69  SEQ ID NO: 5376 tcatgtctcagcctcctca 2620 2639 1 5 SEQ ID NO: 5299 gaggtgccaaggaaatgag 51 70  SEQ ID NO: 5377 ctcatgtctcagcctcctc 2619 2638 1 5 SEQ ID NO: 5300 gggaaagataaagccpacc 487 506  SEQ ID NO: 5378 ggtcgcctggcttattccc 1295 1314 1 5 SEQ ID NO: 5301 ttttcctcatcaagttgtt 598 617  SEQ ID NO: 5379 aacattttcgaaaagaaaa 2982 3001 1 5 SEQ ID NO: 5302 ctttcagccacatccacag 651 670  SEQ ID NO: 5380 ctgtggactctggagaaag 773 792 1 5 SEQ ID NO: 5303 tggactctggagaaagccc 776 795  SEQ ID NO: 5381 gggctggctctcaactcca 884 903 1 5 SEQ ID NO: 5304 agcctcctcaagaacctgg 848 867  SEQ ID NO: 5382 ccagattcttccactggct 2107 2128 1 5 SEQ ID NO: 5305 ggcctggggctggctctca 878 897  SEQ ID NO: 5383 tgagccaccgcaccgggcc 2801 2820 1 5 SEQ ID NO: 5306 gagctcactcccactggaa 1439 1458  SEQ ID NO: 5384 ttccaggtagggccagctc 1678 1895 1 5 SEQ ID NO: 5307 agctaatgaagctattgag 1572 1591  SEQ ID NO: 5385 ctcagcctcctcagtagct 2626 2645 1 5 SEQ ID NO: 5308 gctaatgaagctattgaga 1573 1592  SEQ ID NO: 5386 tctcagcctcctcagtagc 2625 2644 1 5 SEQ ID NO: 5309 ctaaatggctttaattata 1854 1873  SEQ ID NO: 5387 tatatttttagaattttag 2883 2702 1 5 SEQ ID NO: 5310 ctgcttttctttttttttc 2509 2528  SEQ ID NO: 5388 gaaaaatatatatgtgcag 2998 3015 1 5 SEQ ID NO: 5311 caatcaccaccaagcctgg 0 19  SEQ ID NO: 5389 ccagaatgggtccacattg 812 831 1 4 SEQ ID NO: 5312 agcctggaataactgcaag 12 31  SEQ ID NO: 5390 cttggatttctgaatggct 1987 2006 1 4 SEQ ID NO: 5313 gttccatcttcaggaagct 220 239  SEQ ID NO: 5391 agctcactcccactggaac 1440 1459 1 4 SEQ ID NO: 5314 tggtgggttttggatactg 326 345  SEQ ID NO: 5392 cagtcctcccaccctacca 2425 2444 1 4 SEQ ID NO: 5315 acctgtgagactggaccag 392 411  SEQ ID NO: 5393 ctggagaaagcccagaggt 782 801 1 4 SEQ ID NO: 5316 gctgttacagaaactttca 638 657  SEQ ID NO: 5394 tgaatggtcttctgccagc 1474 1493 1 4 SEQ ID NO: 5317 cagcatctataatgccag 666 685  SEQ ID NO: 5395 ctgggtgtagacctcctgt 758 777 1 4 SEQ ID NO: 5318 gggtgtagacctcctgtgg 760 779  SEQ ID NO: 5396 ccacattgacaccacaccc 823 842 1 4 SEQ ID NO: 5319 ggtgtagacctcctgtgga 761 780  SEQ ID NO: 5397 tccacattgacaccacacc 822 841 1 4 SEQ ID NO: 5320 gtgtagacctcctgtggac 762 781  SEQ ID NO: 5398 gtccacattgacaccacac 821 840 1 4 SEQ ID NO: 5321 gacctcctgtggactctgg 767 786  SEQ ID NO: 5399 ccagatattgcactaggtc 2014 2033 1 4 SEQ ID NO: 5322 cctgggcacgctctttggc 862 881  SEQ ID NO: 5400 gccagctcacaagcccegg 1687 1706 1 4 SEQ ID NO: 5323 ctgggcacgctttggcc 863 882  SEQ ID NO: 5401 ggccagctcacaagcccag 1888 1705 1 4 SEQ ID NO: 5324 ctggtcttctacgtcttat 1028 1047  SEQ ID NO: 5402 acaaaagcaagacttccag 1663 1882 1 4 SEQ ID NO: 5325 agagtgcggtagtgcccct 1056 1075  SEQ ID NO: 5403 agggccaggattcctctct 2229 2248 1 4 SEQ ID NO: 5326 tgggcactggtatttggag 1217 1236  SEQ ID NO: 5404 ctcccactggaacagccca 1446 1465 1 4 SEQ ID NO: 5327 gaattaaatcacggatggc 1267 1286  SEQ ID NO: 5405 gccaaccaagagcacattc 2311 2330 1 4 SEQ ID NO: 5328 tgttgctagaagttgggtt 1598 1617  SEQ ID NO: 5406 aaccatcctgctcataaca 2967 2986 1 4 SEQ ID NO: 5329 aggagctcgaatctgate 1764 1783  SEQ ID NO: 5407 tatcacattacatcatcct 2063 2082 1 4 SEQ ID NO: 5330 taaatggctttaattatat 1855 1874  SEQ ID NO: 5408 atatatgtgcagtatttta 3003 3022 1 4 SEQ ID NO: 5331 aaaatgacaaggggagggc 2215 2234  SEQ ID NO: 5409 gccctccttgcctgttttt 2817 2836 1 4 SEQ ID NO: 5332 ttaaaggaaaagtcaacat 2330 2349  SEQ ID NO: 5410 atgtgcagtattttattaa 3007 3026 1 4 SEQ ID NO: 5333 acatcttctctcttttttt 2345 2364  SEQ ID NO: 5411 aaaagaaaaatatatatgt 2992 3011 1 4 SEQ ID NO: 5334 ttctacgtcctcttcccca 197 216  SEQ ID NO: 5412 tgggccagccgcacaagaa 1116 1135 1 3 SEQ ID NO: 5335 tgggtagctgtattggag 257 276  SEQ ID NO: 5413 ctcccactggaacagccca 1446 1485 1 3 SEQ ID NO: 5336 gctgtgattggagactggc 263 282  SEQ ID NO: 5414 gccatgccatgggcacagc 423 442 1 3 SEQ ID NO: 5337 cacttccgtgcccctgata 358 377  SEQ ID NO: 5415 tatcacccaggctggagtg 2548 2567 1 3 SEQ ID NO: 5338 acatctactctriccatct 484 483  SEQ ID NO: 5416 agatgggatttcatcatgt 2705 2724 1 3 SEQ ID NO: 5339 ctactctttccatctttca 468 487  SEQ ID NO: 5417 tgaatactatcacaagtag 1419 1438 1 3 SEQ ID NO: 5340 agataaagccgacctacag 492 511  SEQ ID NO: 5418 ctgtttttcaatctcatct 2828 2847 1 3 SEQ ID NO: 5341 tgtgcagctgaatgtctgt 553 572  SEQ ID NO: 5419 acagaaactttcagccaca 644 863 1 3 SEQ ID NO: 5342 atgtctgtctgtcacgaat 564 583  SEQ ID NO: 5420 attcaggtatagctgacat 2038 2057 1 3 SEQ ID NO: 5343 ctgtcacgaatctaccttg 572 591  SEQ ID NO: 5421 caaggtgctaggattacag 2779 2798 1 3 SEQ ID NO: 5344 atcaagttgttgctggagt 606 625  SEQ ID NO: 5422 actcctgacctcaagtgat 2742 2761 1 3 SEQ ID NO: 5345 cagaaactttcagccacat 645 664  SEQ ID NO: 5423 atgtttcaattaggctctg 2185 2204 1 3 SEQ ID NO: 5346 actttcagccacatccaca 650 669  SEQ ID NO: 5424 tgtggcgtatcatgcaagt 1818 1837 1 3 SEQ ID NO: 5347 atgccagcctcaagaaata 678 697  SEQ ID NO: 5425 tattttttttactgtgcat 1950 1969 1 3 SEQ ID NO: 5348 agaaatattttctcattac 690 709  SEQ ID NO: 5426 gtaaatatgactcctttct 2283 2302 1 3 SEQ ID NO: 5349 gaaatattttctcattacc 691 710  SEQ ID NO: 5427 ggtaaatatgactcctttc 2282 2301 1 3 SEQ ID NO: 5350 tgctgctcaaagggactggg 744 763  SEQ ID NO: 5428 cccaagccaaaccaagagca 2306 2325 1 3 SEQ ID NO: 5351 cctgtggactctggagaaa 772 791  SEQ ID NO: 5429 tttcatcatgttggccagg 2713 2732 1 3 SEQ ID NO: 5352 ggagaaagcccagaggtgg 784 803  SEQ ID NO: 5430 ccaccgcaccgggccctccc 2805 2824 1 3 SEQ ID NO: 5353 ttgaaacccccatcccaag 1004 1023  SEQ ID NO: 5431 cttgaattcctuggctcaa 2405 2424 1 3 SEQ ID NO: 5354 cagatggaggtgccatatc 1351 1370  SEQ ID NO: 5432 gatatgcagagtatttctg 2847 2866 1 3 SEQ ID NO: 5355 ggagctcactcccactgga 1438 1457  SEQ ID NO: 5433 tccacctgccttggcctcc  2760 2779 1 3 SEQ ID NO: 5356 ttgggtaatgtttttgaaa 1553 1572  SEQ ID NO: 5434 tttctctcccaagccaa 2297 2316 1 3 SEQ ID NO: 5357 gaagttgggttgttctgga 1606 1625  SEQ ID NO: 5435 tccaccccactggatcttc 2131 2150 1 3 SEQ ID NO: 5358 aaaagaaggctgcctaagg 1785 1804  SEQ ID NO: 5436 ccttgcctgcttttctttt 2503 2522 1 3 SEQ ID NO: 5359 aaagaaggctgcctaagga 1786 1805  SEQ ID NO: 5437 tccttgcctgcttttcttt 2502 2521 1 3 SEQ ID NO: 5360 aagaaggctgcctaaggag 1787 1806  SEQ ID NO: 5438 ctccttgcctgcttttctt 2501 2520 1 3 SEQ ID NO: 5361 agaaggctgcctaaggagg 1788 1807  SEQ ID NO: 5439 cctccttgcctgcttttct 2500 2519 1 3 SEQ ID NO: 5362 atttccttggatttctgaa 1982 2001  SEQ ID NO: 5440 ttcaattaggctctgaaat 2189 2208 1 3 SEQ ID NO: 5363 tccttataagcccagctct 2081 2100  SEQ ID NO: 5441 agegcacattcttaaagga 2319 2338 1 3 SEQ ID NO: 5364 ataagcccagctctgcttt 2086 2105  SEQ ID NO: 5442 aaagctgaagcctatttat 2889 2908 1 3 SEQ ID NO: 5365 ggccaggattcctctca 2231 2250  SEQ ID NO: 5443 tgagccaccgcaccgggcc 2801 2820 1 3 SEQ ID NO: 5366 gccaactcctccttgcctg 2493 2512  SEQ ID NO: 5444 caggctggagtggagtggc 2555 2574 1 3 SEQ ID NO: 5367 ttttttttctttttttgag 2519 2538  SEQ ID NO: 5445 ctcataacatctttgaaaa 2977 2996 1 3 SEQ ID NO: 5368 ccggcgtgcaccaccatgc 2652 2671  SEQ ID NO: 5446 gcatgagccaccgcaccgg 2798 2817 1 3

TABLE 11 Selected palindromic sequences from rat glucose-6-phosphatase Start End Start  End Source Index Index Match Index Index # B SEQ ID NO: 5447 ctgactattacagcaacag 301 320 SEQ ID NO: 5471 ctgtggctgaaactttcag 598 617 1 6 SEQ ID NO: 5448 ctcttggggttggggctgg 831 850 SEQ ID NO: 5472 ccagcatgtaccgcaagag 859 878 1 6 SEQ ID NO: 5449 tgcaaaggagaactgcgca 879 898 SEQ ID NO: 5473 tgcgaccgtcccctttgca 1019 1038 1 6 SEQ ID NO: 5450 cctcgggccatgccatggg 376 395 SEQ ID NO: 5474 cccagtgtggggccagagg 1171 1190 1 5 SEQ ID NO: 5451 ttgagcaaaccatatgcaa 1478 1497 SEQ ID NO: 5475 ttgcagagtgtgcttacaa 2057 2076 1 5 SEQ ID NO: 5452 cagcttcctgaggaccaa 2 21 SEQ ID NO: 5476 ttggtgtctgtgatcgctg 123 142 1 4 SEQ ID NO: 5453 ggtaccaaggaggaaggat 13 32 SEQ ID NO: 5477 atccagtcgactcgctacc 66 85 1 4 SEQ ID NO: 5454 ctccacgactttgggatcc 51 70 SEQ ID NO: 5478 ggatcgggaggagggggag 1448 1467 1 4 SEQ ID NO: 5455 caggactggtttgtcttgg 108 127 SEQ ID NO: 5479 ccaagcccgactgtgcctg 2018 2037 1 4 SEQ ID NO: 5456 cttctatgtcctattccc 155 174 SEQ ID NO: 5480 gggacagacacacaagaag 1076 1095 1 4 SEQ ID NO: 5457 ttctatgtcctctttccca 156 175 SEQ ID NO: 5481 tgggacagacacacaagaa 1075 1094 1 4 SEQ ID NO: 5458 tggttccacattcaagaga 177 196 SEQ ID NO: 5482 tctcaataatgatagacca 1549 1568 1 4 SEQ ID NO: 5459 tgcctctgataaaacagtt 325 344 SEQ ID NO: 5483 aactctgagatcttgggca 1868 1887 1 4 SEQ ID NO: 5460 agcccggetcdgggacag 1064 1083 SEQ ID NO: 5484 ctgtcctccagcctgggct 2034 2053 1 4 SEQ ID NO: 5461 agtctctgacacaagtcag 1111 1130 SEQ ID NO: 5485 ctgaatggtaatggtgact 1659 1678 1 4 SEQ ID NO: 5462 aaaaaggtgaatttttaaa 1237 1256 SEQ ID NO: 5486 tttattaaaacgacatttt 2201 2220 1 4 SEQ ID NO: 5463 acactctcaataatgatag 1545 1564 SEQ ID NO: 5487 ctatgaatgatgcctgtgt 2121 2140 1 4 SEQ ID NO: 5464 aaagaatgaacgtgctcca 37 56 SEQ ID NO: 5488 tggacctcctgtggacttt 724 743 1 3 SEQ ID NO: 5465 ctttgggatccagtcgact 59 78 SEQ ID NO: 5489 agtcagcggccgtgcaaag 1124 1143 1 3 SEQ ID NO: 5466 gtgatcgctgacctcagga 132 151 SEQ ID NO: 5490 tcctctctccaaaggtcac 1911 1930 1 3 SEQ ID NO: 5467 ggaacgccttctatgtcct 148 167 SEQ ID NO: 5491 aggactcatcactgcttcc 1748 1767 1 3 SEQ ID NO: 5468 gactgtgggcatcaatctc 194 213 SEQ ID NO: 5492 gagactggaccagggagtc 357 376 1 3 SEQ ID NO: 5469 ggacactgactattacagc 296 315 SEQ ID NO: 5493 gctgaacgtctgtctgtcc 518 537 1 3 SEQ ID NO: 5470 aagcccccgtcccagattg 966 985 SEQ ID NO: 5494 caattgtttgctggtgctt 1833 1852 1 3

TABLE 12 Selected palindromic sequences from human 5-catenin   Start End   Start End Source Index Index Match Index Index # B SEQ ID NO: 5495 agcagcttcagtccccgcc 70 89 SEQ ID NO: 5542 ggcgacatatgcagctgct 2152 2171 1 5 SEQ ID NO: 5496 ccattctggtgccactacc 304 323 SEQ ID NO: 5543 ggtatggaccccatgatgg 2387 2406 1 5 SEQ ID NO: 5497 tccttctctgagtggtaaa 328 347 SEQ ID NO: 5544 tttattacatcaagaagga 985 1004 1 5 SEQ ID NO: 5498 tctgagtggtaaaggcaat 334 353 SEQ ID NO: 5545 attgtacgtaccatgcaga 791 810 1 5 SEQ ID NO: 5499 cagagggtacgagctgcta 473 492 SEQ ID NO: 5546 tagctgcaggggtcctctg 2037 2056 1 5 SEQ ID NO: 5500 ctaaatgacgaggaccagg 677 696 SEQ ID NO: 5547 cctgtaaatcatcctttag 2539 2558 1 5 SEQ ID NO: 5501 taaatgacgaggaccaggt 678 697 SEQ ID NO: 5548 acctgtaaatcatccttta 2538 2557 1 5 SEQ ID NO: 5502 gtcctgtatgagtgggaac 383 402 SEQ ID NO: 5549 gttccgaatgtctgaggac 2176 2195 2 4 SEQ ID NO: 5503 cccagcgccgteccaccat 1839 1858 SEQ ID NO: 5550 atgggctgccagatctggg 2451 2470 2 4 SEQ ID NO: 5504 tcccctgagggtatttgaa 143 162 SEQ ID NO: 5551 ttcacatcctagctcggga 1929 1948 1 4 SEQ ID NO: 5505 gggtatttgaagtatacca 151 170 SEQ ID NO: 5552 tggttaagctcttacaccc 1680 1699 1 4 SEQ ID NO: 5506 gctgttagtcactggcagc 260 279 SEQ ID NO: 5553 gctgcctccaggtgacagc 2494 2513 1 4 SEQ ID NO: 5507 gtcctgtatgagtgggaac 383 402 SEQ ID NO: 5554 gttcgccttcactatggac 1652 1671 1 4 SEQ ID NO: 5508 tcctgtatgagtgggaaca 384 403 SEQ ID NO: 5555 tgttccgaatgtctgagga 2175 2194 1 4 SEQ ID NO: 5509 gtatgcaatgactcgagct 454 473 SEQ ID NO: 5556 agctggcctggtttgatac 2517 2536 1 4 SEQ ID NO: 5510 gtccagcgtttggctgaac 563 582 SEQ ID NO: 5557 gttcgccttcactatggac 1652 1671 1 4 SEQ ID NO: 5511 tatcaagatgatgcagaac 623 642 SEQ ID NO: 5558 gttcgtgcacatcaggata 1820 1839 1 4 SEQ ID NO: 5512 tatggtccatcagctttct 718 737 SEQ ID NO: 5559 agaaagcaagctcatcata 1126 1145 1 4 SEQ ID NO: 5513 ccctggtgaaaatgcttgg 915 934 SEQ ID NO: 5560 ccaaagagtagctgcaggg  2029 2048 1 4 SEQ ID NO: 5514 agctttaggacttcacctg 1291 1310 SEQ ID NO: 5561 caggtgacagcaatcagct 2502 2521 1 4 SEQ ID NO: 5515 ggaatctttcagatgctac 1356 1375 SEQ ID NO: 5562 gcagctgctgttttgttcc 2162 2181 1 4 SEQ ID NO: 5516 tgtccttcgggctggtgac 1549 1568 SEQ ID NO: 5563 gtcatctgaccagccgaca 1605 1624 1 4 SEQ ID NO: 5517 cacagctcctctgacagag 2107 2126 SEQ ID NO: 5564 ctctaggaatgaaggtgtg 2134 2153 1 4 SEQ ID NO: 5518 ccagacagaaaagcggctg 245 264 SEQ ID NO: 5565 cagctcgttgtaccgctgg 828 847 2 3 SEQ ID NO: 5519 cagcagcgttggcccggcc 4 23 SEQ ID NO: 5566 ggccaccaccctggtgctg 2420 2439 1 3 SEQ ID NO: 5520 aggtctgaggagcagcttc 60 79 SEQ ID NO: 5567 gaagaggatgtggatacct 359 378 1 3 SEQ ID NO: 5521 actgatttgaaaatccagc 174 193 SEQ ID NO: 5568 gctgatattgatggacagt 437 456 1 3 SEQ ID NO: 5522 ctgatttgatggagttgga 213 232 SEQ ID NO: 5569 tccaggtgacagcaatcag 2500 2519 1 3 SEQ ID NO: 5523 ccagacagaaaagcggctg 245 264 SEQ ID NO: 5570 cagcaacagtcttacctgg 275 294 1 3 SEQ ID NO: 5524 acagctccttctctgagtg 323 342 SEQ ID NO: 5571 cactgagcctgccatctgt 1579 1598 1 3 SEQ ID NO: 5525 tggatacctcccaagtcct 369 388 SEQ ID NO: 5572 aggactaaataccattcca 1972 1991 1 3 SEQ ID NO: 5526 tcaagaacaagtagctgat 424 443 SEQ ID NO: 5573 atcagctggcctggtttga 2514 2533 1 3 SEQ ID NO: 5527 agctcagagggtacgagct 469 488 SEQ ID NO: 5574 agctggtggaatgcaagct 1276 1295 1 3 SEQ ID NO: 5528 gcatgcagatcccatctac 516 535 SEQ ID NO: 5575 gtagaagctggtggaatgc 1271 1290 1 3 SEQ ID NO: 5529 ccacacgtgcaatccctga 645 664 SEQ ID NO: 5576 tcagatgatataaatgtgg 1430 1449 1 3 SEQ ID NO: 5530 cacacgtgcaatccctgaa 646 665 SEQ ID NO: 5577 ttcagatgatataaatgtg 1429 1448 1 3 SEQ ID NO: 5531 ggaccttgcataacctttc 846 865 SEQ ID NO: 5578 gaaatcttgccctttgtcc 1743 1762 1 3 SEQ ID NO: 5532 ctccacaaccttttattac 974 993 SEQ ID NO: 5579 gtaaatcatcctttaggag 2542 2561 1 3 SEQ ID NO: 5533 cagagtgctgaaggtgcta 1222 1241 SEQ ID NO: 5580 tagctgcaggggtcctctg 2037 2056 1 3 SEQ ID NO: 5534 ggactctcaggaatctttc 1347 1366 SEQ ID NO: 5581 gaaatcttgccctttgtcc 1743 1762 1 3 SEQ ID NO: 5535 tgatataaatgtggtcacc 1435 1454 SEQ ID NO: 5582 ggtgacagggaagacatca 1562 1581 1 3 SEQ ID NO: 5536 cccagcgccgtacgtccat 1839 1858 SEQ ID NO: 5583 atggccaggatgccttggg 2370 2389 1 3 SEQ ID No: 5537 gtccatgggtgggacacag 1852 1871 SEQ ID NO: 5584 ctgtgaacttgctcaggac 2053 2072 1 3 SEQ ID NO: 5538 ttgtaccggagcccttcac 1915 1934 SEQ ID NO: 5585 gtgaacttgctcaggacaa 2055 2074 1 3 SEQ ID NO: 5539 ttgttatcagaggactaaa 1962 1981 SEQ ID NO: 5586 tttaggagtaacaatacaa 2553 2572 1 3 SEQ ID NO: 5540 gaagctattgaagctgagg 2084 2103 SEQ ID NO: 5587 cctctgacagagttcttc 2114 2133 1 3 SEQ ID NO: 5541 tcagaacagagccaatggc 2247 2266 SEQ ID NO: 5588 gccaccaccctggtgctga 2421 2440 1 3

TABLE 13  Selected palindromic sequences from human hepatitis C virus (HCV) Start End  Start  End  Source  Index Index Match Index Index # B SEQ ID NO: 5589 cagcacctgggtgctggta 5314 5333 SEQ ID NO: 6135 taccatcacccagctgctg 6196 6215 1 9 SEQ ID NO: 5590 aactcgtccggatgcccgg 1682 1701 SEQ ID NO: 6136 ccgggcagcgggtcgagtt 8202 8221 1 8 SEQ ID NO: 5591 cgctgctgggtagcgctca 1049 1068 SEQ ID NO: 6137 tgagagcgacgccgcagcg 6151 6170 1 7 SEQ ID NO: 5592 ctccggatcccacaagccg 1352 1371 SEQ ID NO: 6138 cggcatgtgggcccgggag 6053 6072 1 7 SEQ ID NO: 5593 tgtaacatcgggggggteg 2048 2067 SEQ ID NO: 6139 cgacccctcccacattaca 6871 6890 1 7 SEQ ID NO: 5594 gtaacategggggggtegg 2049 2068 SEQ ID NO: 6140 ccgacccctcccacattac 6870 6889 1 7 SEQ ID NO: 5595 cagccaccaagcaggcgga 5556 5575 SEQ ID NO: 6141 tccggctggttcgttgctg 9254 9273 1 7 SEQ ID NO: 5596 ctcaccacccagaacaccc 5744 5763 SEQ ID NO: 6142 gggtgtgcacggtgttgag 6291 6310 1 7 SEQ ID NO: 5597 ccagccttaccatcaccca 6189 6208 SEQ ID NO: 6143 tgggcgctggtatcgctgg 5832 5851 1 7 SEQ ID NO: 5598 ctacgccgtgttccggctc 6249 6268 SEQ ID NO: 6144 gagcccgaaccggacgtag 6830 6849 1 7 SEQ ID NO: 5599 tacgccgtgttccggctcg 6250 6269 SEQ ID NO: 6145 cgagcccgaaccggacgta 6829 6848 1 7 SEQ ID NO: 5600 gagttcctggtaaaagcct 8216 8235 SEQ ID NO: 6146 aggctatgactaggtactc 8634 8653 1 7 SEQ ID NO: 5601 atggcggggaactgggcta 1430 1449 SEQ ID NO: 6147 tagcgcattttcactccat 9019 9038 2 6 SEQ ID NO: 5602 aaccaaacgtaacaccaac 370 389 SEQ ID NO: 6148 gttgccgctaccttaggtt 4115 4134 1 6 SEQ ID NO: 5603 ggtggtcagatcgttggtg 419 438 SEQ ID NO: 6149 caccagcccgctcaccacc 5734 5753 1 6 SEQ ID NO: 5604 ccttggcccctctatggca 584 603 SEQ ID NO: 6150 tgccaacgtgggtacaagg 6374 6393 1 6 SEQ ID NO: 5605 taccccggccacgcgtcag 1265 1284 SEQ ID NO: 6151 ctgacgactagctgcggta 8465 8484 1 6 SEQ ID NO: 5606 gggcacgctgcccgcctca 1508 1527 SEQ ID NO: 6152 tgagacgacgaccgtgccc 4759 4778 1 6 SEQ ID NO: 5607 ctgcaatgactccctccag 1624 1643 SEQ ID NO: 6153 ctggtggccctcaatgcag 2594 2613 1 6 SEQ ID NO: 5608 aaccgatcgtctcggcaac 1897 1916 SEQ ID NO: 6154 gttgccgctaccttaggtt 4115 4134 1 6 SEQ ID NO: 5609 gtgcggggcccccccgtgt 2032 2051 SEQ ID NO: 6155 acaccacgggcccctgcac 6537 6556 1 6 SEQ ID NO: 5610 atgtggggggcgtggagca 2238 2257 SEQ ID NO: 6156 tgctcaatgtcctacacat 7610 7629 1 6 SEQ ID NO: 5611 ggagagcgttgcaacttgg 2288 2307 SEQ ID NO: 6157 ccaagctcaaactcactcc 9207 9226 1 6 SEQ ID NO: 5612 cgtccgttgccggagcgca 2613 2632 SEQ ID NO: 6158 tgcgagcccgaaccggacg 6827 6846 1 6 SEQ ID NO: 5613 gtctggcattattgacctt 2817 2836 SEQ ID NO: 6159 aaggtcacctttgacagac 7763 7782 1 6 SEQ ID NO: 5614 tctttgatatcaccaaact 2997 3016 SEQ ID NO: 6160 agttcgatgaaatggaaga 5454 5473 1 6 SEQ ID NO: 5615 cttctgattgccatactcg 3014 3033 SEQ ID NO: 6161 cgagcaattcaagcagaag 5518 5537 1 6 SEQ ID NO: 5616 gcggcgtgtggggacatca 3314 3333 SEQ ID NO: 6162 tgatcacgccatgcgccgc 7641 7660 1 6 SEQ ID NO: 5617 gggacatcatcctgggcct 3324 3343 SEQ ID NO: 6163 aggcggtggattttgtccc 3915 3934 1 6 SEQ ID NO: 5618 gggcgtcttccgggccgct 3874 3893 SEQ ID NO: 6164 agcggcacggcgaccgccc 7439 7458 1 6 SEQ ID NO: 5619 ggcgtcttccgggccgctg 3875 3894 SEQ ID NO: 6165 cagcggcacggcgaccgcc 7438 7457 1 6 SEQ ID NO: 5620 gcgtcttccgggccgctgt 3876 3895 SEQ ID NO: 6166 acaggtgccctgatcacgc 7631 7650 1 6 SEQ ID NO: 5621 gtccccggtcttcacagac 3961 3980 SEQ ID NO: 6167 gtcttggaagaacccggac 7252 7271 1 6 SEQ ID NO: 5622 catcaggactggggtaagg 4174 4193 SEQ ID NO: 6168 ccttcctcaagccgtgatg 8155 8174 1 6 SEQ ID NO: 5623 ccgacggtggttgctccgg 4245 4264 SEQ ID NO: 6169 ccgggggaacggccctcgg 4853 4872 1 6 SEQ ID NO: 5624 ggggggaaggcacctcatt 4501 4520 SEQ ID NO: 6170 aatgttgtgacttggcccc 8334 8353 1 6 SEQ ID NO: 5625 ccgagcaattcaagcagaa 5517 5536 SEQ ID NO: 6171 ttctgattgccatactcgg 3015 3034 1 6 SEQ ID NO: 5626 agatgaaggcaaaggcgtc 7821 7840 SEQ ID NO: 6172 gacgaccttgtcgttatct 8564 8583 1 6 SEQ ID NO: 5627 cccctagggggcgctgcca 767 786 SEQ ID NO: 6173 tggccggcgccccccgggg 3674 3693 1 6 SEQ ID NO: 5628 ctcccggcctagttggggc 646 665 SEQ ID NO: 6174 gcccccccttgagggggag 7519 7538 2 5 SEQ ID NO: 5629 ttccgctcgtcggcggccc 750 769 SEQ ID NO: 6175 gggcaaaggacgtccggaa 7923 7942 2 5 SEQ ID NO: 5630 cccctagggggcgctgcca 767 786 SEQ ID NO: 6176 tggcgggggcccactgggg 1383 1402 2 5 SEQ ID NO: 5631 gccccgccggcatgcgaca 1222 1241 SEQ ID NO: 6177 tgtcccagggggggagggc 9147 9166 2 5 SEQ ID NO: 5632 aggacgaccgggtcctttc 178 197 SEQ ID NO: 6178 gaaaaaggacggttgtcct 7341 7360 1 5 SEQ ID NO: 5633 ggacgaccgggtcctttct 179 198 SEQ ID NO: 6179 agaaaaaggacggttgtcc 7340 7359 1 5 SEQ ID NO: 5634 aaaaccaaacgtaacacca 368 387 SEQ ID NO: 6180 tggtttttttttttttttt 9443 9462 1 5 SEQ ID NO: 5635 caaccgccgcccacaggac 385 404 SEQ ID NO: 6181 gtcctgaacccgtctgttg 4100 4119 1 5 SEQ ID NO: 5636 cggtggtcagatcgttggt 418 437 SEQ ID NO: 6182 accattgagacgacgaccg 4754 4773 1 5 SEQ ID NO: 5637 acctgttgccgcgcagggg 444 463 SEQ ID NO: 6183 ccccggccacgcgtcaggt 1267 1286 1 5 SEQ ID NO: 5633 tgccgcgcaggggccccag 450 469 SEQ ID NO: 6184 ctgggcgcgctgacgggca 3164 3183 1 5 SEQ ID NO: 5639 gggccccaggttgggtgtg 460 479 SEQ ID NO: 6185 cacagcctgtctcgtgccc 9296 9315 1 5 SEQ ID NO: 5640 gttggggccccacggaccc 657 676 SEQ ID NO: 6186 gggtgggtagccgcccaac 5783 5802 1 5 SEQ ID NO: 5641 ttggggccccacggacccc 658 677 SEQ ID NO: 6187 ggggtgggtagccgcccaa 5782 5801 1 5 SEQ ID NO: 5642 tggggccccacggaccccc 659 678 SEQ ID NO: 6188 gggggtgggtagccgccca 5781 5800 1 5 SEQ ID NO: 5643 cctcacatgcggcctcgcc 715 734 SEQ ID NO: 6189 ggcggggcgacaatagagg 3774 3793 1 5 SEQ ID NO: 5644 cacatgcggcctcgccgac 718 737 SEQ ID NO: 6190 gtcgtcggagtcgtgtgtg 6020 6039 1 5 SEQ ID NO: 5645 tccgctcgtcggcggcccc 751 770 SEQ ID NO: 6191 ggggcaaaggacgtccgga 7922 7941 1 5 SEQ ID NO: 5646 ggcgctgccagggccttgg 776 795 SEQ ID NO: 6192 ccaagccacagtgtgcgcc 5110 5129 1 5 SEQ ID NO: 5647 ccatgtcacgaacgactgc 943 962 SEQ ID NO: 6193 gcagcaacacgtggcatgg 6498 6517 1 5 SEQ ID NO: 5648 gtgccctgcgttcgggagg 1019 1038 SEQ ID NO: 6194 cctcacaacgggggggcac 1495 1514 1 5 SEQ ID NO: 5649 tgccctgcgttcgggaggg 1020 1039 SEQ ID NO: 6195 ccctcacaacgggggggca 1494 1513 1 5 SEQ ID NO: 5650 gccctgcgttcgggagggt 1021 1040 SEQ ID NO: 6196 accctcacaacgggggggc 1493 1512 1 5 SEQ ID NO: 5651 aggaatgctaccatcccca 1085 1101 SEQ ID NO: 6197 tgggcatcggcacagtcct 4323 4342 1 5 SEQ ID NO: 5652 tccccactacgacaatacg 1098 1117 SEQ ID NO: 6198 cgtattcccagatttggga 8092 8111 1 5 SEQ ID NO: 5653 atacgacaccacgtcgatt 1112 1131 SEQ ID NO: 6199 aatcaatgctgtagcgtat 4576 4595 1 5 SEQ ID NO: 5654 atttgctcgttggggcggc 1128 1147 SEQ ID NO: 6200 gccgccacttgcggcaaat 9164 9183 1 5 SEQ ID NO: 5655 ccttctcgccccgccggca 1215 1234 SEQ ID NO: 6201 tgccaacgtgggtacaagg 637 6393 1 5 SEQ ID NO: 5656 accccggccacgcgtcagg 1266 1285 SEQ ID NO: 6202 cctgccgcggttaccgggt 6340 6359 1 5 SEQ ID NO: 5657 gccctcgtagtgtcgcagt 1331 1350 SEQ ID NO: 6203 actgcgccggcatgcgggc 6046 6065 1 5 SEQ ID NO: 5658 gccgtctcagagaatccag 1558 1577 SEQ ID NO: 6204 ctggtatcgctggtgcggc 5838 5857 1 5 SEQ ID NO: 5659 ctgaactgcaatgactccc 1619 1638 SEQ ID NO: 6205 gggacagatcggagctcag 2313 2332 1 5 SEQ ID NO: 5660 agactgggtttcttgccgc 1641 1660 SEQ ID NO: 6206 gcggcgagcctacgagtct 8609 8628 1 5 SEQ ID NO: 5661 tcgtccggatgcccggagc 1685 1704 SEQ ID NO: 6207 gctccgggggcgcttacga 4257 4276 1 5 SEQ ID NO: 5662 ccagggatggggtcctatc 1738 1757 SEQ ID NO: 6208 gataacttcccctacctgg 5084 5103 1 5 SEQ ID NO: 5663 gacaaccgatcgtctcggc 1894 1913 SEQ ID NO: 6209 gccgcggttaccgggtgtc 6343 6362 1 5 SEQ ID NO: 5664 caagacgtgcggggccccc 2026 2045 SEQ ID NO: 6210 ggggtctcccccctccttg 6919 6938 1 5 SEQ ID NO: 5665 acgtgcggggcccccccgt 2030 2049 SEQ ID NO: 6211 acgggcgcccccattacgt 4202 4221 1 5 SEQ ID NO: 5666 ccggaagcaccccgaggcc 2101 2120 SEQ ID NO: 6212 ggccgctgtatgcacccgg 3886 3905 1 5 SEQ ID NO: 5667 aggccacgtactcaaaatg 2115 2134 SEQ ID NO: 6213 cattatgtccaaatggcct 3137 3156 1 5 SEQ ID NO: 5668 tgtatgtggggggcgtgga 2235 2254 SEQ ID NO: 6214 tccaagtggcccatctaca 4011 4030 1 5 SEQ ID NO: 5669 gagtggcaggttctgccct 2354 2373 SEQ ID NO: 6215 agggcaggggtggcgactc 3400 3419 1 5 SEQ ID NO: 5670 tcctttgcaatcaaatggg 2474 2493 SEQ ID NO: 6216 cccaccttatgggcaagga 8861 8880 1 5 SEQ ID NO: 5671 agcccaggccgaggccgcc 2566 2585 SEQ ID NO: 6217 ggcgtccacagtcaaggct 7834 7853 1 5 SEQ ID NO: 5672 ggcggcatatgctttctat 2698 2717 SEQ ID NO: 6218 atagaagaagcctgccgcc 7865 7884 1 5 SEQ ID NO: 5673 gcggcatatgctttctatg 2699 2718 SEQ ID NO: 6219 catagaagaagcctgccgc 7864 7883 1 5 SEQ ID NO: 5674 cggcatatgctttctatgg 2700 2719 SEQ ID NO: 6220 ccatagaagaagcctgccg 7863 7882 1 5 SEQ ID NO: 5675 tgcatgtgtgggttccccc 2913 2932 SEQ ID NO: 6221 ggggggacggcatcatgca 6402 6421 1 5 SEQ ID NO: 5676 cccccctcaacgtccgggg 2928 2947 SEQ ID NO: 6222 ccccaatcgatgaacgggg 9376 9395 1 5 SEQ ID NO: 5677 gggcaggggtggcgactcc 3401 3420 SEQ ID NO: 6223 ggaggccgcaagccagccc 8066 8085 1 5 SEQ ID NO: 5678 atgttggactgtctaccat 3574 3593 SEQ ID NO: 6224 atggtaccgaccctaacat 4158 4177 1 5 SEQ ID NO: 5679 tgttggactgtctaccatg 3575 3594 SEQ ID NO: 6225 catggtaccgaccctaaca 4157 4176 1 5 SEQ ID NO: 5680 cgttccctgacaccatgca 3695 3714 SEQ ID NO: 6226 tgcacgatgctcgtgaacg 8543 8562 1 5 SEQ ID NO: 5681 acaccatgcacctgtggca 3704 3723 SEQ ID NO: 6227 tgccgcggttaccgggtgt 6342 6361 1 5 SEQ ID NO: 5682 caccatgcacctgtggcag 3705 3724 SEQ ID NO: 6228 ctgccgcggttaccgggtg 6341 6360 1 5 SEQ ID NO: 5683 ggcatcggcacagtcctgg 4325 4344 SEQ ID NO: 6229 ccaggattgcccgtttgcc 4979 4998 1 5 SEQ ID NO: 5684 aagcggagacggctggagc 4347 4366 SEQ ID NO: 6230 gctccccccagcgctgctt 5804 5823 1 5 SEQ ID NO: 5685 ggagcgcggcttgtcgtgc 4361 4380 SEQ ID NO: 6231 gcacggcgaccgcccctcc 7443 7462 1 5 SEQ ID NO: 5686 cgaagccatcaagggggga 4489 4508 SEQ ID NO: 6232 tccccccagcgctgcttcg 5806 5825 1 5 SEQ ID NO: 5687 tggaagtgtctcatacggc 5165 5184 SEQ ID NO: 6233 gccggattacaatcctcca 7225 7244 1 5 SEQ ID NO: 5688 gggtgctggtaggcggagt 5322 5341 SEQ ID NO: 6234 actcgcgatcccaccaccc 8765 8784 1 5 SEQ ID NO: 5689 gtgggtaggatcatcttgt 5390 5409 SEQ ID NO: 6235 acaacatggtctacgccac 7713 7732 1 5 SEQ ID NO: 5690 cgccgagcaattcaagcag 5515 5534 SEQ ID NO: 6236 ctgcacgccttccccggcg 6550 6569 1 5 SEQ ID NO: 5691 tggagtccaagtggcgagc 5592 5611 SEQ ID NO: 6237 gctcctcatacggattcca 8175 8194 1 5 SEQ ID NO: 5692 tggcgagctttggagacct 5603 5622 SEQ ID NO: 6238 aggtgccctgatcacgcca 7633 7652 1 5 SEQ ID NO: 5693 gcccgctcaccacccagaa 5739 5758 SEQ ID NO: 6239 ttctggcgggctatggggc 5895 5914 1 5 SEQ ID NO: 5694 tgagcgactccaagacccg 6306 6325 SEQ ID NO: 6240 caggctataaaatcgctca 8363 8382 1 5 SEQ ID NO: 5695 atgtcaaaaacggttccat 6456 6475 SEQ ID NO: 6241 atggtaccgaccctaacat 4158 4177 1 5 SEQ ID NO: 5696 ccgaaaacctgcagcaaca 6488 6507 SEQ ID NO: 6242 tgttcctccaatgtgtcgg 8708 8727 1 5 SEQ ID NO: 5697 ggcgccaaactattccaag 6565 6584 SEQ ID NO: 6243 cttgaaagcctctgccgcc 8500 8519 1 5 SEQ ID NO: 5698 gccctccttgagggcgaca 6967 6986 SEQ ID NO: 6244 tgtctcctacttgaagggc 3814 3833 1 5 SEQ ID NO: 5699 cacccgcgtggagtcggag 7078 7097 SEQ ID NO: 6245 ctccggtggtacacgggtg 7278 7297 1 5 SEQ ID NO: 5700 ggagggggatgagaatgaa 7130 7157 SEQ ID NO: 6246 ttcatgctgtgcctactcc 9326 9345 1 5 SEQ ID NO: 5701 gcggcgatacccatatggg 7202 7221 SEQ ID NO: 6247 cccagggggggagggccgc 9150 9169 1 5 SEQ ID NO: 5702 ttgccacctgtcaaggccc 7301 7320 SEQ ID NO: 6248 gggccgccacttgcggcaa 9162 9181 1 5 SEQ ID NO: 5703 cccccccttgagggggagc 7520 7539 SEQ ID NO: 6249 gctcccggcctagttgggg 645 664 1 5 SEQ ID NO: 5704 ctgctgctcaatgtcctac 7606 7625 SEQ ID NO: 6250 gtaggactggcaggggcag 4809 4828 1 5 SEQ ID NO: 5705 catggacaggtgccctgat 7626 7645 SEQ ID NO: 6251 atcattgaacgactccatg 8996 9015 1 5 SEQ ID NO: 5706 atggacaggtgccctgatc 7627 7646 SEQ ID NO: 6252 gatcattgaacgactccat 8995 9014 1 5 SEQ ID NO: 5707 ggctatgactaggtactcc 9635 8654 SEQ ID NO: 6253 ggagcaacttgaaaaagcc 8920 8939 1 5 SEQ ID NO: 5708 caccatagatcactcccct 27 46 SEQ ID NO: 6254 agggccttggcacatggtg 785 804 2 4 SEQ ID NO: 5709 agctgttcaccttctcgcc 1206 1225 SEQ ID NO: 6255 ggcgtgctgacgactagct 8459 8478 2 4 SEQ ID NO: 5710 ctgcaatgactccctccag 1624 1643 SEQ ID NO: 6256 ctggtgcggctgttggcag 5847 5866 2 4 SEQ ID NO: 5711 atgtggggggcgtggagca 2238 2257 SEQ ID NO: 6257 tgctgcgccatcacaacat 7701 7720 2 4 SEQ ID NO: 5712 tggggacatcatcctgggc 3322 3341 SEQ ID NO: 6258 gcccaactcgctcccccca 5795 5814 2 4 SEQ ID NO: 5713 gggacatcatcctgggcct 3324 3343 SEQ ID NO: 6259 aggcaggagataacttccc 5076 5095 2 4 SEQ ID NO: 5714 gggagatactcctggggcc 3366 3385 SEQ ID NO: 6260 ggcccctgcacgccttccc 6545 6564 2 4 SEQ ID NO: 5715 atgttggactgtctaccat 3574 3593 SEQ ID NO: 6261 atggtctacgccacgacat 7711 87737 2 4 SEQ ID NO: 5716 ccagccttaccatcaccca 6189 6208 SEQ ID NO: 6262 tgggtacaagggagtctgg 6382 6401 2 4 SEQ ID NO: 5717 gccctccttgagggcgaca 6967 6986 SEQ ID NO: 6263 tgtcccagggggggagggc 9117 9166 2 4 SEQ ID NO: 5718 ccagcccccgattgggggc 1 20 SEQ ID NO: 6264 gcccgagggcagggcctgg 550 569 1 4 SEQ ID NO: 5719 accatagatcactcccctg 28 47 SEQ ID NO: 6265 cagggccttggcacatggt 794 803 1 4 SEQ ID NO: 5720 atgagtgtcgtgcagcctc 95 114 SEQ ID NO: 6266 gaggccgcgatgccatcat 2946 2965 1 4 SEQ ID NO: 5721 gtgcagcctccaggacccc 104 123 SEQ ID NO: 6267 gggggacggcatcatgcac 6403 6422 1 4 SEQ ID NO: 5722 tgcagcctccaggaccccc 105 124 SEQ ID NO: 6268 ggggggacggcatcatgca 6402 6421 1 4 SEQ ID NO: 5723 ccaggaccccccctcccgg 113 132 SEQ ID NO: 6269 ccggctggttcgttgctgg 9255 9274 1 4 SEQ ID NO: 5724 accccccctcccgggagag 118 137 SEQ ID NO: 6270 ctctcatgccaacgtgggt 6368 6387 1 4 SEQ ID NO: 5725 ccccctcccgggagagcca 121 140 SEQ ID NO: 6271 tggcaatgagggcatgggg 598 617 1 4 SEQ ID NO: 5726 agactgctagccgagtagt 243 262 SEQ ID NO: 6272 actatgcggtccccggtct 3953 3972 1 4 SEQ ID NO: 5727 agccgagtagtgttgggtc 251 270 SEQ ID NO: 6273 gaccaggatctcgtcggct 3656 3675 1 4 SEQ ID NO: 5728 ggtgcttgcgagtgccccg 299 318 SEQ ID NO: 6274 cggggccttggttgacacc 2139 2158 1 4 SEQ ID NO: 5729 gcgagtgccccgggaggtc 306 325 SEQ ID NO: 6275 gacccccggcgtaggtcgc 671 690 1 4 SEQ ID NO: 5730 accgtgcaccatgagcacg 331 350 SEQ ID NO: 6276 cgtgcaatacctgtacggt 2437 2456 1 4 SEQ ID NO: 5731 cccgggcggtggtcagatc 412 431 SEQ ID NO: 6277 gatcacgcatactcccggg 997 1016 1 4 SEQ ID NO: 5732 gccgcgcaggggccccagg 451 470 SEQ ID NO: 6278 cctgcacgccttccccggc 6549 6568 1 4 SEQ ID NO: 5733 accccgtggaaggcgacag 511 530 SEQ ID NO: 6279 ctgtatgcacccggggggt 3891 3910 1 4 SEQ ID NO: 5734 ccccgtggaaggcgacagc 512 531 SEQ ID NO: 6280 gctgtatgcacccgggggg 3890 3909 1 4 SEQ ID NO: 5735 agcctatccccaaggctcg 528 547 SEQ ID NO: 6281 cgagggcagggcctgggct 553 572 1 4 SEQ ID NO: 5736 ctatccccaaggctcgccg 531 550 SEQ ID NO: 6282 cggctgtcgttcccgatag 5418 5437 1 4 SEQ ID NO: 5737 tatccccaaggctcgccgg 532 551 SEQ ID NO: 6283 ccggctgtcgttcccgata 5417 5436 1 4 SEQ ID NO: 5738 cgggtatccttggcccctc 577 596 SEQ ID NO: 6284 gaggccgcaagccagcccg 8067 8086 1 4 SEQ ID NO: 5739 gcatggggtgggcaggatg 609 628 SEQ ID NO: 6285 catcgataccctcacatgc 706 725 1 4 SEQ ID NO: 5740 tcctgtcaccccgcggctc 630 649 SEQ ID NO: 6286 gagctgcaaagctccagga 8523 8542 1 4 SEQ ID NO: 5741 gggccccacggacccccgg 661 680 SEQ ID NO: 6287 ccggccgcatatgcggccc 4064 4083 1 4 SEQ ID NO: 5742 ggccccacggacccccggc 662 681 SEQ ID NO: 6288 gccggccgcatatgcggcc 4063 4082 1 4 SEQ ID NO: 5743 cggcctcgccgacctcatg 724 743 SEQ ID NO: 6289 catgaggatcatcgggccg 6472 6491 1 4 SEQ ID NO: 5744 ggcctcgccgacctcatgg 725 744 SEQ ID NO: 6290 ccatgaggatcatcgggcc 6471 6490 1 4 SEQ ID NO: 5745 ggccccctagggggcgctg 764 783 SEQ ID NO: 6291 cagctccgaattgtcggcc 7414 7433 1 4 SEQ ID NO: 5746 tggcacatggtgtccgggt 792 811 SEQ ID NO: 6292 acccacgctgcacgggcca 5188 5207 1 4 SEQ ID NO: 5747 cttcctcttggctctgctg 868 887 SEQ ID NO: 6293 cagcataggtcttgggaag 5863 5882 1 4 SEQ ID NO: 5748 catgtcacgaacgactgct 944 963 SEQ ID NO: 6294 agcagtgctcacttccatg 6847 6866 1 4 SEQ ID NO: 5749 gaggcggcggacttgatca 983 1002 SEQ ID NO: 6295 tgatggcattcacagcctc 5712 5731 1 4 SEQ ID NO: 5750 catccccactacgacaata 1096 1115 SEQ ID NO: 6296 tattaccggggtcttgatg 4592 4611 1 4 SEQ ID NO: 5751 gctgttcaccttctcgccc 1207 1226 SEQ ID NO: 6297 gggctgcgtgggaaacagc 8793 8812 1 4 SEQ ID NO: 5752 gccccgccggcatgcgaca 1222 1241 SEQ ID NO: 6298 tgtctcctacttgaagggc 3814 3833 1 4 SEQ ID NO: 5753 tggcctgggacatgatgat 1293 1312 SEQ ID NO: 6299 atcaatttgctccctgcca 5981 6000 1 4 SEQ ID NO: 5754 cacaagccgtcatcgacat 1362 1381 SEQ ID NO: 6300 atgtttgggactgggtgtg 6279 6298 1 4 SEQ ID NO: 5755 agccgtcatcgacatggtg 1366 1385 SEQ ID NO: 6301 caccaagcaggcggaggct 5560 5579 1 4 SEQ ID NO: 5756 ggtggcgggggcccactgg 1381 1400 SEQ ID NO: 6302 ccagggctcaggccccacc 5127 5146 1 4 SEQ ID NO: 5757 gggggcccactggggagtc 1387 1406 SEQ ID NO: 6303 gactaggtactccgccccc 8641 8660 1 4 SEQ ID NO: 5758 atggcggggaactgggcta 1430 1449 SEQ ID NO: 6304 tagcagtgctcacttccat 6846 6865 1 4 SEQ ID NO: 5759 ttgattgtgatgctacttt 1454 1473 SEQ ID NO: 6305 aaagcaagctgcccatcaa 7665 7684 1 4 SEQ ID NO: 5760 caacgggggggcacgctgc 1500 1519 SEQ ID NO: 6306 gcagaaggcgctcgggttg 5530 5549 1 4 SEQ ID NO: 5761 acgctgcccgcctcaccag 1512 1531 SEQ ID NO: 6307 ctggacccgaggagagcgt 2278 2297 1 4 SEQ ID NO: 5762 tcagagaatccagcttata 1564 1583 SEQ ID NO: 6308 tatatcgggggtcccctga 8393 8412 1 4 SEQ ID NO: 5763 accaatggcagttggcaca 1586 1605 SEQ ID NO: 6309 tgtggctcggggccttggt 2132 2151 1 4 SEQ ID NO: 5764 ccaatggcagttggcacat 1587 1606 SEQ ID NO: 6310 atgtggctcggggccttgg 2131 2150 1 4 SEQ ID NO: 5765 gtcctatcacttatgctga 1749 1768 SEQ ID NO: 6311 tcaggactggggtaaggac 4176 4195 1 4 SEQ ID NO: 5766 ctgagcctacaaaagaccc 1764 1783 SEQ ID NO: 6312 gggtggcttcatgcctcag 9063 9082 1 4 SEQ ID NO: 5767 caggtgtgtggtccagtgt 1844 1863 SEQ ID NO: 6313 acactccagttaactcctg 8817 8836 1 4 SEQ ID NO: 5768 tgtggtccagtgtattgct 1850 1869 SEQ ID NO: 6314 agcagggccatcaaccaca 7949 7968 1 4 SEQ ID NO: 5769 gcttcaccccaagtcctgt 1866 1885 SEQ ID NO: 6315 acagcagaggcggctaagc 6887 6906 1 4 SEQ ID NO: 5770 ctgttgtcgtggggacaac 1881 1900 SEQ ID NO: 6316 gttgcaacttggacgacag 2295 2314 1 4 SEQ ID NO: 5771 gccgccgcaaggcaactgg 1972 1991 SEQ ID NO: 6317 ccagttggacttatccggc 9241 9260 1 4 SEQ ID NO: 5772 ggcaactggttcggctgta 1982 2001 SEQ ID NO: 6318 tacacgggtgcccattgcc 7287 7306 1 4 SEQ ID NO: 5773 gcaactggttcggctgtac 1983 2002 SEQ ID NO: 6319 gtacacgggtgcccattgc 7286 7305 1 4 SEQ ID NO: 5774 ccccgtgtaacatcggggg 2043 2062 SEQ ID NO: 6320 ccccaatcgatgaacgggg 9376 9395 1 4 SEQ ID NO: 5775 ggactgcttccggaagcac 2092 2111 SEQ ID NO: 6321 gtgctggtaggcggagtcc 5324 5343 1 4 SEQ ID NO: 5776 gactgcttccggaagcacc 2093 2112 SEQ ID NO: 6322 ggtgctggtaggcggagtc 5323 5342 1 4 SEQ ID NO: 5777 tccggaagcaccccgaggc 2100 2119 SEQ ID NO: 6323 gcctacgagtcttcacgga 3616 8635 1 4 SEQ ID NO: 5778 actcaaaatgtggctcggg 2124 2143 SEQ ID NO: 6324 cccgggcagcgggtcgagt 8201 8220 1 4 SEQ ID NO: 5779 ggccttggttgacacctag 2142 2161 SEQ ID NO: 6325 ctagccggcccaaaaggcc 3611 3630 1 4 SEQ ID NO: 5780 aggagagcgttgcaacttg 2287 2306 SEQ ID NO: 6326 caagccgtgatgggctcct 8162 8181 1 4 SEQ ID NO: 5781 ggacagatcggagctcagc 2314 2333 SEQ ID NO: 6327 gctgggggtcattatgtcc 3128 3147 1 4 SEQ ID NO: 5782 cagatcggagctcagcccg 2317 2336 SEQ ID NO: 6328 cgggtggcccactgctctg 3837 3856 1 4 SEQ ID NO: 5783 ggagctcagcccgctgctg 2323 2342 SEQ ID NO: 6329 cagctgctgaagaggctcc 6206 6225 1 4 SEQ ID NO: 5784 caccctaccggctctgtcc 2383 2402 SEQ ID NO: 6330 ggactgggtgtgcacggtg 6286 6305 1 4 SEQ ID NO: 5785 cggctctgtccactggctt 2391 2410 SEQ ID NO: 6331 aagcaggcggaggctgccg 5564 5583 1 4 SEQ ID NO: 5786 ccatcagaacatcgtggac 2419 2438 SEQ ID NO: 6332 gtccccgtcgagtccatgg 3929 3948 1 4 SEQ ID NO: 5787 ggtcagcggttgtctcctt 2460 2479 SEQ ID NO: 6333 aaggatgattctgatgacc 8875 8894 1 4 SEQ ID NO: 5788 gccgccttagagaacctgg 2579 2598 SEQ ID NO: 6334 ttgggcccacgccggccta 3217 3236 1 4 SEQ ID NO: 5789 gccttagagaacctggtgg 2582 2601 SEQ ID NO: 6335 ccaccaagcaggcggaggc 5559 5578 1 4 SEQ ID NO: 5790 gccggagcgcacggcatcc 2621 2640 SEQ ID NO: 6336 ggattgggcccacgccggc 3214 3233 1 4 SEQ ID NO: 5791 gctgcatcgtgcggaggcg 2786 2805 SEQ ID NO: 6337 cgccacgacatcccgcagc 7726 7745 1 4 SEQ ID NO: 5792 attattgaccttgtcgcca 2824 2843 SEQ ID NO: 6338 tggcaacagacgctctaat 4647 4666 1 4 SEQ ID NO: 5793 tcgccatattacaaggtgt 2837 2856 SEQ ID NO: 6339 acacaatctttcctggcga 3539 3558 1 1 SEQ ID NO: 5794 cgccatattacaaggtgtt 2838 2857 SEQ ID NO: 6340 aacacaatctttcctggcg 3538 3557 1 4 SEQ ID NO: 5795 gtccggggaggccgcgatg 2939 2958 SEQ ID NO: 6341 catcggcacagtcctggac 4327 4346 1 4 SEQ ID NO: 5796 tcaccccactgcgggattg 3201 3220 SEQ ID NO: 6342 caatttaccaatgttgtga 8325 8344 1 4 SEQ ID NO: 5797 ttqqqcccacqccqqccta 3217 3236 SEQ ID NO: 6343 taqqctaqqqqccqtccaa 5221 5240 1 4 SEQ ID NO: 5798 ctacgggaccttgcggtag 3233 3252 SEQ ID NO: 6344 ctactcctactttctgtag 9338 9357 1 4 SEQ ID NO: 5799 cctgtcgtcttctctgaca 3260 3279 SEQ ID NO: 6345 tgtcctacacatggacagg 7617 7636 1 4 SEQ ID NO: 5800 ctgtcgtcttctctgacat 3261 3280 SEQ ID NO: 6346 atgtcctacacatggacag 7616 7635 1 4 SEQ ID NO: 5801 cctggggggcagacaccgc 3297 3316 SEQ ID NO: 6347 gcggggtaggactggcagg 4804 4823 1 4 SEQ ID NO: 5802 gggggcagacaccgcggcg 3301 3320 SEQ ID NO: 6348 cgcccaactcgctcccccc 5794 5813 1 4 SEQ ID NO: 5803 ggcgtgtggggacatcatc 3316 3335 SEQ ID NO: 6349 gatgttattccggtgcgcc 3755 3774 1 4 SEQ ID NO: 5804 tggggccggccgatagtct 3378 3397 SEQ ID NO: 6350 agacgacgaccgtgcccca 4761 4780 1 4 SEQ ID NO: 5805 gaaccaggtcgagggggag 3499 3518 SEQ ID NO: 6351 ccccacctatggcaagttc 4222 4241 1 4 SEQ ID NO: 5806 gagggggaggttcaagtgg 3509 3528 SEQ ID NO: 6352 ccacctgtcaaggcccctc 7304 7323 1 4 SEQ ID NO: 5807 aggcccaatcgcccagatg 3625 3644 SEQ ID NO: 6353 catcccgcagcgcgggcct 7734 7753 1 4 SEQ ID NO: 5808 ggcccaatcgcccagatgt 3626 3645 SEQ ID NO: 6354 acatcccgcagcgcgggcc 7733 7752 1 4 SEQ ID NO: 5809 caggatctcgtcggctggc 3659 3678 SEQ ID NO: 6355 gccaataggccatttcctg 9410 9429 1 4 SEQ ID NO: 5810 aggatctcgtcggctggcc 3660 3679 SEQ ID NO: 6356 ggccaataggccatttcct 9409 9428 1 4 SEQ ID NO: 5811 gccccccggggcgcgttcc 3682 3701 SEQ ID NO: 6357 ggaacctatccagcagggc 7938 7957 1 4 SEQ ID NO: 5812 gcacctgtggcagctcgga 3711 3730 SEQ ID NO: 6358 tccggtggtacacgggtgc 7279 7298 1 4 SEQ ID NO: 5813 ctgtggcagctcggacctt 3715 3734 SEQ ID NO: 6359 aaggcaaaggcgtccacag 7826 7845 1 4 SEQ ID NO: 5814 gcggggcgacaatagaggg 3775 3794 SEQ ID NO: 6360 ccctgcctgggaaccccgc 5682 5701 1 4 SEQ ID NO: 5815 ggagcttgctctcccccag 3792 3811 SEQ ID NO: 6361 ctggttgggtcacagctcc 6806 6825 1 4 SEQ ID NO: 5816 gagcttgctctcccccagg 3793 3812 SEQ ID NO: 6362 cctggttgggtcacagctc 6805 6824 1 4 SEQ ID NO: 5817 acttgaagggctcttcggg 3822 3841 SEQ ID NO: 6363 cccgtggtggagtccaagt 5585 5604 1 4 SEQ ID NO: 5818 tgtccccgttgagtccatg 3928 3947 SEQ ID NO: 6364 catggtctacgccacgaca 7717 7736 1 4 SEQ ID NO: 5819 gaaactactatgcggtccc 3947 3966 SEQ ID NO: 6365 gggaaggcacctcattttc 4504 4523 1 4 SEQ ID NO: 5820 aaactactatgcggtcccc 3948 3967 SEQ ID NO: 6366 ggggggcatatacaggttt 4828 4847 1 4 SEQ ID NO: 5821 ctcccactggcagcggcaa 4032 4051 SEQ ID NO: 6367 ttgccaggaccatctggag 4993 5012 1 4 SEQ ID NO: 5822 ggcgtatatgtctaaagca 4138 4157 SEQ ID NO: 6368 tgctcgccaccgctacgcc 4377 4396 1 4 SEQ ID NO: 5823 gcgtatatgtctaaagcac 4139 4158 SEQ ID NO: 6369 gtgctcgccaccgctacgc 4376 4395 1 4 SEQ ID NO: 5824 tggggtaaggaccattacc 4183 4202 SEQ ID NO: 6370 ggtaaccatgtctccccca 6119 6138 1 4 SEQ ID NO: 5825 accattaccacgggcgccc 4193 4212 SEQ ID NO: 6371 gggcgctggtatcgctggt 5833 5852 1 4 SEQ ID NO: 5826 cgtactccacctatggcaa 4218 4237 SEQ ID NO: 6372 ttgccccaaccagaatacg 8669 8688 1 4 SEQ ID NO: 5827 cagtcctggaccaagcgga 4335 4354 SEQ ID NO: 6373 tccgtgagccgcatgactg 9560 9579 1 4 SEQ ID NO: 5828 aggggggaaggcacctcat 4500 4519 SEQ ID NO: 6374 atgagcggcgaggcgccct 5948 5967 1 4 SEQ ID NO: 5829 cactccaagaagaagtgcg 4526 4545 SEQ ID NO: 6375 cgcatgactgcagagagtg 9569 9588 1 4 SEQ ID NO: 5830 atcaatgctgtagcgtatt 4577 4596 SEQ ID NO: 6376 aatacgacttggagttgat 8682 8701 1 4 SEQ ID NO: 5831 cataccgaccagcggagac 4618 4637 SEQ ID NO: 6377 gtctcccccacgcactatg 6128 6147 1 4 SEQ ID NO: 5832 aggactggcaggggcaggg 4811 4830 SEQ ID NO: 6378 ccctgccatcctctctcct 5992 6011 1 4 SEQ ID NO: 5833 gggaacggccctcgggcat 4857 4876 SEQ ID NO: 6379 atgctcaccgacccctccc 6863 6882 1 4 SEQ ID NO: 5834 cgggcatgttcgattcctc 4869 4888 SEQ ID NO: 6380 gaggccgcaagccagcccg 8067 8086 1 4 SEQ ID NO: 5835 tggtacgagctcacccccg 4922 4941 SEQ ID NO: 6381 cggggacttgccccaacca 8662 8681 1 4 SEQ ID NO: 5836 gggcttacctaaatacacc 4962 4981 SEQ ID NO: 6382 ggtggctccatcttagccc 9518 9537 1 4 SEQ ID NO: 5837 ggcttacctaaatacacca 4963 4982 SEQ ID NO: 6383 tggtggctccatcttagcc 9517 9536 1 4 SEQ ID NO: 5838 gagataacttcccctacct 5082 5101 SEQ ID NO: 6384 aggttggccagggggtctc 6908 6927 1 4 SEQ ID NO: 5839 cccacctccatcgtgggat 5140 5159 SEQ ID NO: 6385 atccaagtttggctatggg 7906 7925 1 4 SEQ ID NO: 5840 catggcatgcatgtcggcc 5278 5297 SEQ ID NO: 6386 ggcctctctgcagatcatg 9596 9615 1 4 SEQ ID NO: 5841 ggccgacctggaagtcgtc 5293 5312 SEQ ID NO: 6387 gacgcccccacattcggcc 7885 7904 1 4 SEQ ID NO: 5842 gccgacctggaagtcgtca 5294 5313 SEQ ID NO: 6388 tgacgcccccacattcggc 7884 7903 1 4 SEQ ID NO: 5843 tggaagtcgtcaccagcac 5301 5320 SEQ ID NO: 6389 gtgcccatgtcaggttcca 6676 6695 1 4 SEQ ID NO: 5844 gcacctgggtgctggtagg 5316 5335 SEQ ID NO: 6390 cctacacatggacaggtgc 7620 7639 1 4 SEQ ID NO: 5845 ggttatcgtgggtaggatc 5383 5402 SEQ ID NO: 6391 gatcatcgggccgaaaacc 6478 6497 1 4 SEQ ID NO: 5846 cccgatagggaagtcctct 5429 5448 SEQ ID NO: 6392 agagcggctttatatcggg 8383 8402 1 4 SEQ ID NO: 5847 tgaaatggaagaatgcgcc 5461 5480 SEQ ID NO: 6393 ggcgcgctcgtggccttca 5924 5943 1 4 SEQ ID NO: 5848 ccaagtggcgagctttgga 5598 5617 SEQ ID NO: 6394 tccattgttagagtcttgg 7240 7259 1 4 SEQ ID NO: 5849 ttcatcagcgggatacagt 5645 5664 SEQ ID NO: 6395 actgcacgatgctcgtgaa 8541 8560 1 4 SEQ ID NO: 5850 agcgggcttatccaccctg 5668 5687 SEQ ID NO: 6396 caggggtggctggcgcgct 5913 5932 1 4 SEQ ID NO: 5851 ccagcccgctcaccaccca 5736 5755 SEQ ID NO: 6397 tgggcgctggtatcgctgg 5832 5851 1 4 SEQ ID NO: 5852 gtgggcgctggtatcgctg 5831 5850 SEQ ID NO: 6398 cagcagggccatcaaccac 7948 7967 1 4 SEQ ID NO: 5853 ggaaggtgctagtggacat 5877 5896 SEQ ID NO: 6399 atgtggtctccacccttcc 8142 8161 1 4 SEQ ID NO: 5854 ggtcatgagcggcgaggcg 5944 5963 SEQ ID NO: 6400 cgcccctcctgaccagacc 7453 7472 1 4 SEQ ID NO: 5855 catgtgggcccgggagagg 6056 6075 SEQ ID NO: 6401 cctccttgagggcgacatg 6969 6988 1 4 SEQ ID NO: 5856 atgtgggcccgggagaggg 6057 6076 SEQ ID NO: 6402 ccctccttgagggcgacat 6968 6987 1 4 SEQ ID NO: 5857 ggggccgtgcagtggatga 6074 6093 SEQ ID NO: 6403 tcatgctcctctatgcccc 7505 7524 1 4 SEQ ID NO: 5858 gcgttcgcttcgcggggta 6104 6123 SEQ ID NO: 6404 taccaccacgagcttacgc 2751 2770 1 4 SEQ ID NO: 5859 ggggtaaccatgtctcccc 6117 6136 SEQ ID NO: 6405 gggggagccgggggacccc 7531 7550 1 4 SEQ ID NO: 5860 catcacccagctgctgaag 6199 6218 SEQ ID NO: 6406 cttcgagcggagggggatg 7130 7149 1 4 SEQ ID NO: 5861 aggactgttctacgccgtg 6240 6259 SEQ ID NO: 6407 cacggcgaccgcccctcct 7444 7463 1 4 SEQ ID NO: 5862 ttcaagacctggctccagt 6314 6333 SEQ ID NO: 6408 actgcacgatgctcgtgaa 8541 8560 1 4 SEQ ID NO: 5863 ctcctgccgcggttaccgg 6338 6357 SEQ ID NO: 6409 ccgggacgtgcttaaggag 7804 7823 1 4 SEQ ID NO: 5864 caccacgggcccctgcacg 6538 6557 SEQ ID NO: 6410 cgtggaggtcacgcgggtg 6613 6632 1 4 SEQ ID NO: 5865 ggaggtcacgcgggtgggg 6616 6635 SEQ ID NO: 6411 cccctccaataccacctcc 7317 7336 1 4 SEQ ID NO: 5866 gaggtcacgcgggtggggg 6617 6636 SEQ ID NO: 6412 cccctcctgaccagacctc 7455 7474 1 4 SEQ ID NO: 5867 atgtcaggttccagctcct 6682 6701 SEQ ID NO: 6413 aggagatgggcggaaacat 7059 7078 1 4 SEQ ID NO: 5868 atgaaatatccattgcggc 7152 7171 SEQ ID NO: 6414 gccgtgatgggctcctcat 8165 8184 1 4 SEQ ID NO: 5869 ctccattgttagagtcttg 7239 7258 SEQ ID NO: 6415 caagtggcgagctttggag 5599 5618 1 4 SEQ ID NO: 5870 tgcccattgccacctgtca 7295 7314 SEQ ID NO: 6416 tgactaattcaaaagggca 8409 8428 1 4 SEQ ID NO: 5871 accacctccacggagaaaa 7327 7346 SEQ ID NO: 6417 ttttttccctctttatggt 9502 9521 1 4 SEQ ID NO: 5872 ccacctccacggagaaaaa 7328 7347 SEQ ID NO: 6418 ttttccctctttatggtgg 9504 9523 1 4 SEQ ID NO: 5873 acctccacggagaaaaagg 7330 7349 SEQ ID NO: 6419 cctttgacagactgcaggt 7770 7789 1 4 SEQ ID NO: 5874 ggttgtcctgacggactcc 7351 7370 SEQ ID NO: 6420 ggagctcgctaccaaaacc 7390 7409 1 4 SEQ ID NO: 5875 cctgaccagacctccgaca 7460 7479 SEQ ID NO: 6421 tgtcctacacatggacagg 7617 7636 1 4 SEQ ID NO: 5876 agcaagctgcccatcaacg 7667 7686 SEQ ID NO: 6422 cgttgagcaactctttgct 7686 7705 1 4 SEQ ID NO: 5877 ggatgaccattaccgggac 7792 7811 SEQ ID NO: 6423 gtcccagttggacttatcc 9238 9257 1 4 SEQ ID NO: 5878 tggcaaagaatgaggtttt 8028 8047 SEQ ID NO: 6424 aaaaagccctggattgcca 8931 8950 1 4 SEQ ID NO: 5879 ggcaaagaatgaggttttc 8029 8048 SEQ ID NO: 6425 gaaaaagccctggattgcc 8930 8949 1 4 SEQ ID NO: 5880 gggcagcgggtcgagttcc 8204 8223 SEQ ID NO: 6426 ggaagaaagcaagctgccc 7660 7679 1 4 SEQ ID NO: 5881 gactagctgcggtaatacc 8470 8489 SEQ ID NO: 6427 ggtaccgcccttgcgagtc 9091 9110 1 4 SEQ ID NO: 5882 ctcgcgatcccaccacccc 8766 8785 SEQ ID NO: 6428 ggggtaccgcccttgcgag 9089 9108 1 4 SEQ ID NO: 5883 aggatgattctgatgaccc 8876 8895 SEQ ID NO: 6429 gggtcagcggttgtctcct 2459 2478 1 4 SEQ ID NO: 5884 agccacttgacctacctca 8976 8995 SEQ ID NO: 6430 tgagatcaatagggtggct 9052 9071 1 4 SEQ ID NO: 5885 gggtaccgcccttgcgagt 9090 9109 SEQ ID NO: 6431 actcgcgatcccaccaccc 8765 8784 1 4 SEQ ID NO: 5886 ctgcaatgactccctccag 1624 1643 SEQ ID NO: 6432 ctggcgggctatggggcag 5897 5916 3 3 SEQ ID NO: 5887 ccagcccccgattgggggc 1 20 SEQ ID NO: 6433 gcccactggggagtcctgg 1391 1410 2 3 SEQ ID NO: 5888 aaggcgacagcctatcccc 520 539 SEQ ID NO: 6434 gggggtctcccccctcctt 6918 6937 2 3 SEQ ID NO: 5889 ggccccacggacccccggc 662 681 SEQ ID NO: 6435 gccgcaaagctgtcaggcc 4553 4572 2 3 SEQ ID NO: 5890 gaggcggcggacttgatca 983 1002 SEQ ID NO: 6436 tgataacatcatgttcctc 8697 8716 2 3 SEQ ID NO: 5891 ctgcaattgttcgatctac 1249 1268 SEQ ID NO: 6437 gtaggcggagtcctcgcag 5330 5349 2 3 SEQ ID NO: 5892 ctccagactgggtttctcg 1637 1656 SEQ ID NO: 6438 caagtggcgagctttggag 5599 5618 2 3 SEQ ID NO: 5893 tcgtacctgcgtcgcaggt 1830 1849 SEQ ID NO: 6439 acctcagatcattgaacga 8989 9008 2 3 SEQ ID NO: 5894 caagacgtgcggggccccc 2026 2045 SEQ ID NO: 6440 gggggagggccgccacttg 9156 9175 2 3 SEQ ID NO: 5895 aatgctgcatgcaactgga 2264 2283 SEQ ID NO: 6441 tccaggccaataggccatt 9405 9424 2 3 SEQ ID NO: 5896 caccctaccggctctgtcc 2383 2402 SEQ ID NO: 6442 ggactacgtccctccggtg 7267 7286 2 3 SEQ ID NO: 5897 cgccatattacaaggtgtt 2838 2857 SEQ ID NO: 6443 aacagccaccaagcaggcg 5554 5573 2 3 SEQ ID NO: 5898 cgaagccatcaagggggga 4489 4508 SEQ ID NO: 6444 tcccagatttgggagttcg 8097 8116 2 3 SEQ ID NO: 5899 ccagcccgctcaccaccca 5736 5755 SEQ ID NO: 6445 tgggtacaagggagtctgg 6382 6401 2 3 SEQ ID NO: 5900 ggctatgactaggtactcc 8635 8654 SEQ ID NO: 6446 ggagacatatatcacagcc 9284 9303 2 3 SEQ ID NO: 5901 ctccaccatagateactcc 24 43 SEQ ID NO: 6447 ggagacatcgggccaggag 9111 9130 1 3 SEQ ID NO: 5902 tccaccatagatcactccc 25 44 SEQ ID NO: 6448 gggagttcgatgaaatgga 5451 5470 1 3 SEQ ID NO: 5903 caccatagatcactcccct 27 46 SEQ ID NO: 6449 aggggccccaggttgggtg 458 477 1 3 SEQ ID NO: 5904 tcactcccctgtgaggaac 36 55 SEQ ID NO: 6450 gttctggaggacggcgtga 809 828 1 3 SEQ ID NO: 5905 cgttagtatgagtgtcgtg 88 107 SEQ ID NO: 6451 cacgctgcacgggccaacg 5191 5210 1 3 SEQ ID NO: 5906 tgtcgtgcagcctccagga 100 119 SEQ ID NO: 6452 tcctgttgtcgtggggaca 1879 1898 1 3 SEQ ID NO: 5907 ccccccctcccgggagagc 119 138 SEQ ID NO: 6453 gctcccggcctagttgggg 645 664 1 3 SEQ ID NO: 5908 ggagagccatagtggtctg 131 150 SEQ ID NO: 6454 cagatcattgaacgactcc 8993 9012 1 3 SEQ ID NO: 5909 gagccatagtggtctgcgg 134 153 SEQ ID NO: 6455 ccgctgctgggtagcgctc 1048 1067 1 3 SEQ ID NO: 5910 gtggtctgcggaaccggtg 142 161 SEQ ID NO: 6456 cacccatatagatgcccac 5038 5057 1 3 SEQ ID NO: 5911 agtacaccggaattgccag 161 180 SEQ ID NO: 6457 ctggcgggccttgcctact 1406 1425 1 3 SEQ ID NO: 5912 ggtcctttcttggatcaac 188 207 SEQ ID NO: 6458 gttgagtgacttcaagacc 6304 6323 1 3 SEQ ID NO: 5913 ttcttggatcaacccgctc 194 213 SEQ ID NO: 6459 gagcggagggggatgagaa 7134 7153 1 3 SEQ ID NO: 5914 ctcaatgcctggagatttg 210 229 SEQ ID NO: 6460 caaagactccgacgctgag 7486 7505 1 3 SEQ ID NO: 5915 tgcctggagatttgggcgt 215 234 SEQ ID NO: 6461 acgcggccgccgcaaggca 1967 1986 1 3 SEQ ID NO: 5916 gcctggagatttgggcgtg 216 235 SEQ ID NO: 6462 cacgcggccgccgcaaggc 1966 1985 1 3 SEQ ID NO: 5917 gagatttgggcgtgccccc 221 240 SEQ ID NO: 6463 ggggacaaccgatcgtctc 1891 1910 1 3 SEQ ID NO: 5918 aaaggccttgtggtactgc 273 292 SEQ ID NO: 6464 gcagaagaaggtcaccttt 7756 7775 1 3 SEQ ID NO: 5919 aaggccttgtggtactgcc 274 293 SEQ ID NO: 6465 ggcagaagaaggtcacctt 7755 7774 1 3 SEQ ID NO: 5920 gtggtactgcctgataggg 282 301 SEQ ID NO: 6466 ccctaccggctctgtccac 2385 2404 1 3 SEQ ID NO: 5921 cctgatagggtgcttgcga 291 310 SEQ ID NO: 6467 tcgccggcccgagggcagg 544 563 1 3 SEQ ID NO: 5922 cgagtgccccgggaggtct 307 326 SEQ ID NO: 6468 agacgcagtgtcgcgctcg 4780 4799 1 3 SEQ ID NO: 5923 gccccgggaggtctcgtag 312 331 SEQ ID NO: 6469 ctaccttaggttttggggc 4122 4141 1 3 SEQ ID NO: 5924 ttacctgttgccgcgcagg 442 461 SEQ ID NO: 6470 cctgcgttcgggagggtaa 1023 1042 1 3 SEQ ID NO: 5925 tacctgttgccgcgcaggg 443 462 SEQ ID NO: 6471 ccctgcgttcgggagggta 1022 1041 1 2 SEQ ID NO: 5926 cctgttgccgcgcaggggc 445 464 SEQ ID NO: 6472 gcccccgaagccagacagg 8348 8367 1 3 SEQ ID NO: 5927 ctgttgccgcgcaggggcc 446 465 SEQ ID NO: 6473 ggcccccgaagccagacag 8347 8366 1 3 SEQ ID NO: 5928 tccgagcggtcgcaacccc 497 516 SEQ ID NO: 6474 ggggcaaaggacgtccgga 7922 7941 1 3 SEQ ID NO: 5929 ggtcgcaaccccgtggaag 504 523 SEQ ID NO: 6475 cttctctgacatggagacc 3268 3287 1 3 SEQ ID NO: 5930 gtcgcaaccccgtggaagg 505 524 SEQ ID NO: 6476 ccttcaccattgagacgac 4749 4768 1 3 SEQ ID NO: 5931 aaggcgacagcctatcccc 520 539 SEQ ID NO: 6477 ggggcgctgccagggcctt 774 793 1 3 SEQ ID NO: 5932 cagcctatccccaaggctc 527 546 SEQ ID NO: 6478 gagcacaggcttaatgctg 2252 2271 1 3 SEQ ID NO: 5933 gagggcagggcctgggctc 554 573 SEQ ID NO: 6479 gagcgtcttcacaggcctc 5020 5039 1 3 SEQ ID NO: 5934 cagggcctgggctcagccc 559 578 SEQ ID NO: 6480 gggcatcggcacagtcctg 4324 4343 1 3 SEQ ID NO: 5935 gggcctgggctcagcccgg 561 580 SEQ ID NO: 6481 ccggccgcatatgcggccc 4064 4083 1 3 SEQ ID NO: 5936 cctgggctcagcccgggta 564 583 SEQ ID NO: 6482 taccgaccctaacatcagg 4162 4181 1 3 SEQ ID NO: 5937 cccctctatggcaatgagg 590 609 SEQ ID NO: 6483 cctcgccgacctcatgggg 727 746 1 3 SEQ ID NO: 5938 gagggcatggggtgggcag 605 624 SEQ ID NO: 6484 ctgcggatctgttttcctc 1180 1199 1 3 SEQ ID NO: 5939 agggcatggggtgggcagg 606 625 SEQ ID NO: 6485 cctgctctttcaccaccct 2370 2389 1 3 SEQ ID NO: 5940 aggatggctcctgtcaccc 622 641 SEQ ID NO: 6486 gggtcagcggttgtctcct 2459 2478 1 3 SEQ ID NO: 5941 gatggctcctgtcaccccg 624 643 SEQ ID NO: 6487 cgggggcgcttacgacatc 4261 4280 1 3 SEQ ID NO: 5942 tgtcaccccgcggctcccg 633 652 SEQ ID NO: 6488 cggggcgcgttccctgaca 3688 3707 1 3 SEQ ID NO: 5943 gtcaccccgcggctcccgg 634 653 SEQ ID NO: 6489 ccggggcgcgttccctgac 3687 3706 1 3 SEQ ID NO: 5944 gcggctcccggcctagttg 642 661 SEQ ID NO: 6490 caacgtccggggaggccgc 2935 2954 1 3 SEQ ID NO: 5945 ctcccggcctagttggggc 646 665 SEQ ID NO: 6491 gccctgtcgaacactggag 4439 4458 1 3 SEQ ID NO: 5946 ataccctcacatgcggcct 711 730 SEQ ID NO: 6492 aggcaacattatcatgtat 8839 8858 1 3 SEQ ID NO: 5947 ttccgctcgtcggcggccc 750 769 SEQ ID NO: 6493 gggcaaagcacatgtggaa 5625 5644 1 3 SEQ ID NO: 5948 cccctagggggcgctgcca 767 786 SEQ ID NO: 6494 tggcaatgagggcatgggg 598 617 1 3 SEQ ID NO: 5949 tgcaacagggaacctgccc 832 851 SEQ ID NO: 6495 gggctcattcgtgcatgca 3092 3111 1 3 SEQ ID NO: 5950 gcgtaacgcgtccggggta 922 941 SEQ ID NO: 6496 taccaccacgagcttacgc 2751 2770 1 3 SEQ ID NO: 5951 tcaagcattgtgtttgagg 968 987 SEQ ID NO: 6497 cctctatgcccccccttga 7512 7531 1 3 SEQ ID NO: 5952 cccacgctcgcggccagga 1070 1089 SEQ ID NO: 6498 tcctgtttaacatcttggg 5763 5782 1 3 SEQ ID NO: 5953 cggccaggaatgctaccat 1080 1099 SEQ ID NO: 6499 atggcatgcatgtcggccg 5279 5298 1 3 SEQ ID NO: 5954 acgacaatacgacaccacg 1106 1125 SEQ ID NO: 6500 cgtggggacaaccgatcgt 1888 1907 1 3 SEQ ID NO: 5955 gggcggctgctctctgctc 1140 1159 SEQ ID NO: 6501 gagcaacttgaaaaagccc 8921 8940 1 3 SEQ ID NO: 5956 cgtgggggacctctgcgga 1168 1187 SEQ ID NO: 6502 tccgttgccggagcgcacg 2615 2634 1 3 SEQ ID NO: 5957 agctgttcaccttctcgcc 1206 1225 SEQ ID NO: 6503 ggcgacaatagagggagct 3779 3798 1 3 SEQ ID NO: 5958 ctgttcaccttctcgcccc 1208 1227 SEQ ID NO: 6504 ggggagacatatatcacag 9282 9301 1 3 SEQ ID NO: 5959 ctgcaattgttcgatctac 1249 1268 SEQ ID NO: 6505 gtaggactggcaggggcag 4809 4828 1 3 SEQ ID NO: 5961 atctaccccggccacgcgt 1262 1281 SEQ ID NO: 6507 acgccatggaccgggagat 2766 2785 1 3 SEQ ID NO: 5962 cggccacgcgtcaggtcac 1270 1289 SEQ ID NO: 6508 gtgatgctactttttgccg 1460 1479 1 3 SEQ ID NO: 5963 ccgcatggcctgggacatg 1288 1307 SEQ ID NO: 6509 catggaaactactatgcgg 3943 3962 1 3 SEQ ID NO: 5964 cgcagttactccggatccc 1344 1363 SEQ ID NO: 6510 gggaacccaggaggatgcg 8593 8612 1 3 SEQ ID NO: 5965 cccacaagccgtcatcgac 1360 1379 SEQ ID NO: 6511 gtcgtcaccagcacctggg 5306 5325 1 3 SEQ ID NO: 5966 ctggggagtcctggcgggc 1396 1415 SEQ ID NO: 6512 gcccggagcgcatggccag 1695 1714 1 3 SEQ ID NO: 5967 ggcgggccttgcctactat 1408 1427 SEQ ID NO: 6513 atagaagaagcctgccgcc 7865 7884 1 3 SEQ ID NO: 5968 tttgccggcgttgacgggc 1472 1491 SEQ ID NO: 6514 gcccccacattcggccaaa 7888 7907 1 3 SEQ ID NO: 5969 caccctcacaacggggggg 1492 1511 SEQ ID NO: 6515 ccccaatatcgaggaggtg 4420 4439 1 3 SEQ ID NO: 5970 gggggggcacgctgcccgc 1504 1523 SEQ ID NO: 6516 gcggcacggcgaccgcccc 7440 7459 1 3 SEQ ID NO: 5971 ggggcacgctgcccgcctc 1507 1526 SEQ ID NO: 6517 gagggagcttgctctcccc 3789 3808 1 3 SEQ ID NO: 5972 gcccgcctcaccagcgggt 1517 1536 SEQ ID NO: 6518 accctcacaacgggggggc 1493 1512 1 3 SEQ ID NO: 5973 atccagcttataaacacca 1571 1590 SEQ ID NO: 6519 tggttatcgtgggtaggat 5382 5401 1 3 SEQ ID NO: 5974 ctccagactgggtttcttg 1637 1656 SEQ ID NO: 6520 caagcggagacggctggag 4346 4365 1 3 SEQ ID NO: 5975 cccggagcgcatggccagc 1696 1715 SEQ ID NO: 6521 gctgtgggcgtcttccggg 3869 3888 1 3 SEQ ID NO: 5976 ctgccgctccattgacaag 1714 1733 SEQ ID NO: 6522 cttggtacatcaagggcag 2667 2686 1 3 SEQ ID NO: 5977 aagttcgaccagggatggg 1730 1749 SEQ ID NO: 6523 cccaaccagaatacgactt 8673 8692 1 3 SEQ ID NO: 5978 ggggtcctatcacttatgc 1746 1765 SEQ ID NO: 6524 gcatgtgtgggttcccccc 2914 2933 1 3 SEQ ID NO: 5979 ccagaggccttattgctgg 1786 1805 SEQ ID NO: 6525 ccaggatctcgtcggctgg 3658 3677 1 3 SEQ ID NO: 5980 cccacctcaacaatgtggt 1810 1829 SEQ ID NO: 6526 accaagatcatcacctggg 3284 3303 1 3 SEQ ID NO: 5981 tcgtacctgcgtcgcaggt 1830 1849 SEQ ID NO: 6527 accttcaccattgagacga 4748 4767 1 3 SEQ ID NO: 5982 tgcgtcgcaggtgtgtggt 1837 1856 SEQ ID NO: 6528 accatgtctcccccacgca 6123 6142 1 3 SEQ ID NO: 5983 tggggacaaccgatcgtct 1890 1909 SEQ ID NO: 6529 agacgacgaccgtgcccca 4761 4780 1 3 SEQ ID NO: 5984 cagctggggggagaacgat 1924 1943 SEQ ID NO: 6530 atcggagctcagcccgctg 2320 2339 1 3 SEQ ID NO: 5985 cgccgcaaggcaactggtt 1974 1993 SEQ ID NO: 6531 aacccaggaggatgcggcg 8596 8615 1 3 SEQ ID NO: 5986 gccgcaaggcaactggttc 1975 1994 SEQ ID NO: 6532 gaacccaggaggatgcggc 8595 8614 1 3 SEQ ID NO: 5987 ctgtacatggatgaatagc 1996 2015 SEQ ID NO: 6533 gctataaaatcgctcacag 8366 8385 1 3 SEQ ID NO: 5988 tgtacatggatgaatagca 1997 2016 SEQ ID NO: 6534 tgctgctcaatgtcctaca 7607 7626 1 3 SEQ ID NO: 5989 gttcaccaagacgtgcggg 2020 2039 SEQ ID NO: 6535 cccgctcaccacccagaac 5740 5759 1 3 SEQ ID NO: 5990 agacgtgcggggccccccc 2028 2047 SEQ ID NO: 6536 ggggaggttcaagtggtct 3517 3531 1 3 SEQ ID NO: 5991 cccccgtgtaacatcgggg 2042 2061 SEQ ID NO: 6537 ccccaatcgatgaacgggg 9376 9395 1 3 SEQ ID NO: 5992 taacaccttgacctgcccc 2071 2090 SEQ ID NO: 6538 ggggacgaccttgtcgtta 8561 8580 1 3 SEQ ID NO: 5993 ggctctggcactacccctg 2184 2203 SEQ ID NO: 6539 caggaggatgcggcgagcc 8600 8619 1 3 SEQ ID NO: 5994 tgcactgtcaacttctcca 2201 2220 SEQ ID NO: 6540 tggatggggtgcggttgca 6717 6736 1 3 SEQ ID NO: 5995 caggcttaatgctgcatgc 2257 2276 SEQ ID NO: 6541 gcatcatgcacaccacctg 6411 6430 1 3 SEQ ID NO: 5996 aatgctgcatgcaactgga 2264 2283 SEQ ID NO: 6542 tccatggtcttagcgcatt 9009 9028 1 3 SEQ ID NO: 5997 ctgcatgcaactggacccg 2268 2287 SEQ ID NO: 6543 cgggaccttgcggtagcag 3236 3255 1 3 SEQ ID NO: 5998 caactggacccgaggagag 2275 2294 SEQ ID NO: 6544 ctcttacgggatgaggttg 6761 6780 1 3 SEQ ID NO: 5999 gacagggacagatcggagc 2309 2328 SEQ ID NO: 6545 gctctcccccaggcctgtc 3799 3818 1 3 SEQ ID NO: 6000 gacagatcggagctcagcc 2315 2334 SEQ ID NO: 6546 ggctggagcgcggcttgtc 4357 4376 1 3 SEQ ID NO: 6001 acagatcggagctcagccc 2316 2335 SEQ ID NO: 6547 gggccaacgcccctgctgt 5201 5220 1 3 SEQ ID NO: 6002 actggcttgatccacctcc 2402 2421 SEQ ID NO: 6548 ggagagggggccgtgcagt 6068 6087 1 3 SEQ ID NO: 6003 ggctcgatccacctccatc 2405 2424 SEQ ID NO: 6549 gatgatgctgctgatagcc 2551 2570 1 3 SEQ ID NO: 6004 gtcagcggttgtctccttt 2461 2480 SEQ ID NO: 6550 aaaggacggttgtcctgac 7344 7363 1 3 SEQ ID NO: 6005 gagtatgtcgtgttgcttt 2492 2511 SEQ ID NO: 6551 aaagaccaagctcaaactc 9202 9221 1 3 SEQ ID NO: 6006 tgtggatgatgctgctgat 2547 2566 SEQ ID NO: 6552 atcactgatggcattcaca 5707 5726 1 3 SEQ ID NO: 6007 ccgaggccgccttagagaa 2593 2593 SEQ ID NO: 6553 ttctgattgccatactegg 3015 3034 1 3 SEQ ID NO: 6008 agaacctggtggccctcaa 2589 2608 SEQ ID NO: 6554 ttgatatcaccaaacttct 3000 3019 1 3 SEQ ID NO: 6009 tacatcaagggcaggctgg 2672 2691 SEQ ID NO: 6555 ccagatgtacactaatgta 3637 3656 1 3 SEQ ID NO: 6010 caagggcaggctggtccct 2677 2696 SEQ ID NO: 6556 aggggtaggcatctacttg 9355 9374 1 3 SEQ ID NO: 6011 gcatggccgctgctcctgc 2720 2739 SEQ ID NO: 6557 gcagtgctcacttccatgc 6848 6867 1 3 SEQ ID NO: 6012 catggccgctgctcctgct 2721 2740 SEQ ID NO: 6558 agcagtgctcacttccatg 6847 6866 1 3 SEQ ID NO: 6013 gccgctgctcctgctcctc 2725 2744 SEQ ID NO: 6559 gagggccgccacttgcggc 9160 9179 1 3 SEQ ID NO: 6014 ggagatggctgcatcgtgc 2779 2798 SEQ ID NO: 6560 gcacggcgaccgcccctcc 7443 7462 1 3 SEQ ID NO: 6015 atggctgcatcgtgcggag 2783 2802 SEQ ID NO: 6561 ctccaggccaataggccat 9404 9423 1 3 SEQ ID NO: 6016 ggcgcggtttttgtgggcc 2801 2820 SEQ ID NO: 6562 gaccattaccacgggcgcc 4192 4211 1 3 SEQ ID NO: 6017 tcttatcaccagagctgag 2887 2906 SEQ ID NO: 6563 ctcacaggccgggacaaga 3482 3501 1 3 SEQ ID NO: 6018 gtgtgggttccccccctca 2918 2937 SEQ ID NO: 6564 tgaggtcaccctcacacac 5242 5261 1 3 SEQ ID NO: 6019 tccccccctcaacgtccgg 2926 2945 SEQ ID NO: 6565 ccggctcgtggctgaggga 6261 6280 1 3 SEQ ID NO: 6020 ctcaacgtccggggaggcc 2933 2952 SEQ ID NO: 6566 ggcctgttactccattgag 8959 8978 1 3 SEQ ID NO: 6021 accaaacttctgattgcca 3008 3027 SEQ ID NO: 6567 tggctctctacgatgtggt 8130 8149 1 3 SEQ ID NO: 6022 caaacttctgattgccata 3010 3029 SEQ ID NO: 6568 tatgacacccgctgttttg 8267 8286 1 3 SEQ ID NO: 6023 ggaccgctcatggtgctcc 3032 3051 SEQ ID NO: 6569 ggagatcctgcggaagtcc 7171 7190 1 3 SEQ ID NO: 6024 gaccgctcatggtgctcca 3033 3052 SEQ ID NO: 6570 tggaaactactatgcggtc 3945 3964 1 3 SEQ ID NO: 6025 atgcatgttagtgcggaaa 3106 3125 SEQ ID NO: 6571 tttctgtaggggtaggcat 9348 9367 1 3 SEQ ID NO: 6026 ttatgtccaaatggccttc 3139 3158 SEQ ID NO: 6572 gaagccagacaggctataa 3354 8373 1 3 SEQ ID NO: 6027 ccaaatggccttcatgaga 3145 3164 SEQ ID NO: 6573 tctcagcgacgggtcttgg 7552 7571 1 3 SEQ ID NO: 6028 ccttcatgagactgggcgc 3153 3172 SEQ ID NO: 6574 gcgctcgtggccttcaagg 5927 5946 1 3 SEQ ID NO: 6029 ccttgcggtagcagtggag 3241 3260 SEQ ID NO: 6575 ctccgcccgaaggggaagg 3349 3368 1 3 SEQ ID NO: 6030 tgtcgtcttctctgacatg 3262 3281 SEQ ID NO: 6576 catggtctacgccacgaca 7717 7736 1 3 SEQ ID NO: 6031 tggggggcagacaccgcgg 3299 3318 SEQ ID NO: 6577 ccgccttatcgtattccca 8083 8102 1 3 SEQ ID NO: 6032 ggggggcagacaccgcggc 3300 3319 SEQ ID NO: 6578 gccgcccaactcgctcccc 5792 5811 1 3 SEQ ID NO: 6033 gtggggacatcatcctggg 3321 3340 SEQ ID NO: 6579 cccatctacacgctcccac 4020 4039 1 3 SEQ ID NO: 6034 tggggacatcatcctgggc 3322 3341 SEQ ID NO: 6580 gcccatctacacgctccca 4019 4038 1 3 SEQ ID NO: 6035 ggggacatcatcctgggcc 3323 3342 SEQ ID NO: 6581 ggccagggggtctcccccc 6913 6932 1 3 SEQ ID NO: 6036 acctgtctccgcccgaagg 3343 3362 SEQ ID NO: 6582 cctttgacagactgcaggt 7770 7789 1 3 SEQ ID NO: 6037 tgtctccgcccgaagggga 3346 3365 SEQ ID NO: 6583 tccccggtcttcacagaca 3962 3981 1 3 SEQ ID NO: 6038 gggagatactcctggggcc 3366 3385 SEQ ID NO: 6584 ggcccatctacacgctccc 4018 4037 1 3 SEQ ID NO: 6039 ctcccaacagacccggggc 3439 3458 SEQ ID NO: 6585 gcccccccttgagggggag 7519 7538 1 3 SEQ ID NO: 6040 tccaccgcaacacaatctt 3530 3549 SEQ ID NO: 6586 aagaggctccaccagtgga 6215 6234 1 3 SEQ ID NO: 6041 cacaatctttcctggcgac 3540 3559 SEQ ID NO: 6587 gtcgtcggagtcgtgtgtg 6020 6039 1 3 SEQ ID NO: 6042 ggctggccggcgccccccg 3671 3690 SEQ ID NO: 6588 cgggttgttgcaaacagcc 5542 5561 1 3 SEQ ID NO: 6043 ccccggggcgcgttccctg 3685 3704 SEQ ID NO: 6589 caggtttgtaactccgggg 4840 4859 1 3 SEQ ID NO: 6044 tccctgacaccatgcacct 3698 3717 SEQ ID NO: 6590 aggtcacgcgggtggggga 6618 6637 1 3 SEQ ID NO: 6045 ttccggtgcgccggcgggg 3762 3781 SEQ ID NO: 6591 ccccgttgagtccatggaa 3931 3950 1 3 SEQ ID NO: 6046 ctcccccaggcctgtctcc 3802 3821 SEQ ID NO: 6592 ggagacatcgggccaggag 9111 9130 1 3 SEQ ID NO: 6047 gggggttgcaaaggcggtg 3904 3923 SEQ ID NO: 6593 caccctgcctgggaacccc 5680 5699 1 3 SEQ ID NO: 6048 tttgtccccgttgagtcca 3926 3945 SEQ ID NO: 6594 tggagaccttctgggcaaa 5613 5632 1 3 SEQ ID NO: 6049 ccgtaccgcaaacattcca 3996 4015 SEQ ID NO: 6595 tggattgccaaatctacgg 8940 8959 1 3 SEQ ID NO: 6050 caagtggcccatctacacg 4013 4032 SEQ ID NO: 6596 cgtgggtaggatcatcttg 5389 5408 1 3 SEQ ID NO: 6051 cacgctcccactggcagcg 4028 4047 SEQ ID NO: 6597 cgctgcttcggctttcgtg 5815 5834 1 3 SEQ ID NO: 6052 ccgcatatgcggcccaagg 4068 4087 SEQ ID NO: 6598 ccttcaaggtcatgagcgg 5937 5956 1 3 SEQ ID NO: 6053 cgtatatgtctaaagcaca 4140 4159 SEQ ID NO: 6599 tgtggaagtgtctcatacg 5963 5182 1 3 SEQ ID NO: 6054 gtatatgtctaaagcacat 4141 4160 SEQ ID NO: 6600 atgtggaagtgtctcatac 5162 5181 1 3 SEQ ID NO: 6055 ggaccattaccacgggcgc 4191 4210 SEQ ID NO: 6601 gcgcgtgtcactcaggtcc 6167 6186 1 3 SEQ ID NO: 6056 cccccattacgtactccac 4209 4228 SEQ ID NO: 6602 gtgggcccgggagaggggg 6059 6078 1 3 SEQ ID NO: 6057 agttccttgccgacggtgg 4236 42bb SEQ ID NO: 6603 ccacagtcaaggctaaact 7839 7858 1 3 SEQ ID NO: 6058 gagacggctggagcgcggc 4352 4371 SEQ ID NO: 6604 gccgggggaccccgatctc 7537 7556 1 3 SEQ ID NO: 6059 caccgctacgcctccagga 4384 4403 SEQ ID NO: 6605 tcctacacatggacaggtg 7619 7638 1 3 SEQ ID NO: 6060 tggagagatccccttctac 4453 4472 SEQ ID NO: 6606 gtagcagtgctcacttcca 6845 6864 1 3 SEQ ID NO: 6061 agccatccccatcgaagcc 4477 4496 SEQ ID NO: 6607 ggctggttcgttgctggct 9257 9276 1 3 SEQ ID NO: 6062 tccccatcgaagccatcaa 4482 4501 SEQ ID NO: 6608 ttgagggggagccggggga 7527 7546 1 3 SEQ ID NO: 6063 ccccatcgaagccatcaag 4483 4502 SEQ ID NO: 6609 cttgagggggagccggggg 7526 7545 1 3 SEQ ID NO: 6064 ggccccggaaccaatgccg 4568 4587 SEQ ID NO: 6610 cagctccgaattgtcggcc 7414 7433 1 3 SEQ ID NO: 6065 gtccgtcataccgaccagc 4612 4631 SEQ ID NO: 6611 gctgagggatgtttgggac 6271 6290 1 3 SEQ ID NO: 6066 gtcataccgaccagcggag 4616 4635 SEQ ID NO: 6612 ctccattgagccacttgac 8968 8987 1 3 SEQ ID NO: 6067 cgggctataccggtgactt 4668 4687 SEQ ID NO: 6613 aagtccaagaagttccccg 7184 7203 1 3 SEQ ID NO: 6068 ctttgattcagtgatcgac 4684 4703 SEQ ID NO: 6614 gtcgagttcctggtaaaag 8213 8232 1 3 SEQ ID NO: 6069 acagtcgacttcagcttgg 4724 4743 SEQ ID NO: 6615 ccaaatctacggggcctgt 8947 8966 1 3 SEQ ID NO: 6070 cttggaccccaccttcacc 4738 4757 SEQ ID NO: 6616 ggtgttgagtgacttcaag 6301 6320 1 3 SEQ ID NO: 6071 gagacgacgaccgtgcccc 4760 4779 SEQ ID NO: 6617 ggggacaaccgatcgtctc 1891 1910 1 3 SEQ ID NO: 6072 ggggcaggaccggcagggg 4806 4825 SEQ ID NO: 6618 ccccccggggacttgcccc 8657 8676 1 3 SEQ ID NO: 6073 gggcatatacaggtttgta 4831 4850 SEQ ID NO: 6619 tacacatggacaggtgccc 7622 7641 1 3 SEQ ID NO: 6074 gggggaacggccctcgggc 4855 4874 SEQ ID NO: 6620 gcccctgcacgccttcccc 6546 6565 1 3 SEQ ID NO: 6075 tgacgcgggctgtgcttgg 4906 4925 SEQ ID NO: 6621 ccaattgacaccaccgtca 8009 8028 1 3 SEQ ID NO: 6076 gacgcgggctgtgcttggt 4907 4926 SEQ ID NO: 6622 accaattgacaccaccgtc 8008 8027 1 3 SEQ ID NO: 6077 tgcttggtacgagctcacc 4918 4937 SEQ ID NO: 6623 ggtgcggctgttggcagca 5849 5868 1 3 SEQ ID NO: 6078 tgcccacttcctgtcccag 5050 5069 SEQ ID NO: 6624 ctgggcgcgctgacgggca 3164 3183 1 3 SEQ ID NO: 6079 ggtggcataccaagccaca 5101 5120 SEQ ID NO: 6625 tgtgacaccaattgacacc 8002 8021 1 3 SEQ ID NO: 6080 gggctcaggccccacctcc 5130 5149 SEQ ID NO: 6626 ggaggccgcaagccagccc 8066 8085 1 3 SEQ ID NO: 6081 ccatcgtgggatcaaatgt 5147 5166 SEQ ID NO: 6627 acattctggcgggctatgg 5892 5911 1 3 SEQ ID NO: 6082 tcatacggctaaaacccac 5175 5194 SEQ ID NO: 6628 gtggccttcaaggtcatga 5933 5952 1 3 SEQ ID NO: 6083 tgctgtataggctaggggc 5214 5233 SEQ ID NO: 6629 gcccgaaccggacgtagca 6832 6851 1 3 SEQ ID NO: 6084 ccaaatacatcatggcatg 5268 5287 SEQ ID NO: 6630 catgcctcaggaaacttgg 9072 9091 1 3 SEQ ID NO: 6085 ggagtcctcgcagctctgg 5336 5355 SEQ ID NO: 6631 ccagctgtctgcgccctcc 6955 6974 1 3 SEQ ID NO: 6086 gcctgacaacaggcagtgt 5364 5383 SEQ ID NO: 6632 acactccaggccaataggc 9401 9420 1 3 SEQ ID NO: 6087 agccaccaagcaggcggag 5557 5576 SEQ ID NO: 6633 ctccagttaactcctggct 8820 8839 1 3 SEQ ID NO: 6088 catgtggaattteatcage 5635 5654 SEQ ID NO: 6634 gctgcgccatcacaacatg 7702 7721 1 3 SEQ ID NO: 6089 ctctatcaccagcccgctc 5728 5747 SEQ ID NO: 6635 gagccgcatgactgcagag 9565 9584 1 3 SEQ ID NO: 6090 cccagaacaccctcctgtt 5751 5770 SEQ ID NO: 6636 aacatcttgggggggtggg 5771 5790 1 3 SEQ ID NO: 6091 ctcctgtttaacatcttgg 5762 5781 SEQ ID NO: 6637 ccaatcgatgaacggggag 9378 9397 1 3 SEQ ID NO: 6092 ttgggggggtgggtagecg 5777 5796 SEQ ID NO: 6638 cggcgccaaactattccaa 6564 6583 1 3 SEQ ID NO: 6093 tqcttcqqctttcqtqqqc 5818 5837 SEQ ID NO: 6639 qcccqaaccqqacqtaqca 6832 6851 1 3 SEQ ID NO: 6094 tcgtgggcgctggtatcgc 5829 5848 SEQ ID NO: 6640 gcgagcggcgtgctgacga 8453 8472 1 3 SEQ ID NO: 6095 cgctggtgcggctgttggc 5845 5864 SEQ ID NO: 6641 gccacgacatcccgcagcg 7727 7746 1 3 SEQ ID NO: 6096 cggctgttggcagcatagg 5853 5872 SEQ ID NO: 6642 cctagactctttcgagccg 7111 7130 1 3 SEQ ID NO: 6097 ggggcaggggtggctggcg 5909 5928 SEQ ID NO: 6643 cgcccaactcgctcccccc 5794 5813 1 3 SEQ ID NO: 6098 ctggcgcgctcgtggcctt 5922 5941 SEQ ID NO: 6644 aagggaggccgcaagccag 8063 8082 1 3 SEQ ID NO: 6099 tggcgcgctcgtggccttc 5923 5942 SEQ ID NO: 6645 gaagggaggccgcaagcca 8062 8081 1 3 SEQ ID NO: 6100 gagcggcgaggcgccctct 5950 5969 SEQ ID NO: 6646 agagcgtcgtctgctgctc 7596 7615 1 3 SEQ ID NO: 6101 tgggcccgggagagggggc 6060 6079 SEQ ID NO: 6647 gcccatctacacgctccca 4019 4038 1 3 SEQ ID NO: 6102 cggctgatagcgttcgctt 6095 6114 SEQ ID NO: 6648 aagcaggcggaggctgccg 5564 5583 1 3 SEQ ID NO: 6103 gtgcctgagagcgacgccg 6146 6165 SEQ ID NO: 6649 cggccgccgacagcggcac 7428 7447 1 3 SEQ ID NO: 6104 atgaggactgttctacgcc 6237 6256 SEQ ID NO: 6650 ggcggggggacggcatcat 6399 6418 1 3 SEQ ID NO: 6105 gtccaagctcctgccgcgg 6331 6350 SEQ ID NO: 6651 ccgctccgtgtgggaggac 7969 7988 1 3 SEQ ID NO: 6106 acagatcgccggacatgtc 6442 6461 SEQ ID NO: 6652 gacatatatcacagcctgt 9287 9306 1 3 SEQ ID NO: 6107 acgtggcatggaacattcc 6506 6525 SEQ ID NO: 6653 ggaagaacccggactacgt 7257 7276 1 3 SEQ ID NO: 6108 gggcccctgcacgccttcc 6544 6563 SEQ ID NO: 6654 ggaagaaagcaagctgccc 7660 7679 1 3 SEQ ID NO: 6109 agtgcccatgtcaggttcc 6675 6694 SEQ ID NO: 6655 ggaaacagctagacacact 8803 8822 1 3 SEQ ID NO: 6110 tgcccatgtcaggttccag 6677 6696 SEQ ID NO: 6656 ctgggcgcgctgacgggca 3164 3183 1 3 SEQ ID NO: 6111 cagctcctgagtttttcac 6693 6712 SEQ ID NO: 6657 gtgagagcgtcgtctgctg 7593 7612 1 3 SEQ ID NO: 6112 tcacggaggtggatggggt 6708 6727 SEQ ID NO: 6658 acccttcctcaagccgtga 8153 8172 1 3 SEQ ID NO: 6113 cacggaggtggatggggtg 6709 6728 SEQ ID NO: 6659 cacccttcctcaagccgtg 8152 8171 1 3 SEQ ID NO: 6114 gacccctcccacattacag 6872 6891 SEQ ID NO: 6660 ctgttttgactcaacggtc 8278 8297 1 3 SEQ ID NO: 6115 ttggccagggggtctcccc 6911 6930 SEQ ID NO: 6661 ggggtgggtagccgcccaa 5782 5801 1 3 SEQ ID NO: 6116 ccttgagggcgacatgcac 6972 6991 SEQ ID NO: 6662 gtgcttaaggagatgaagg 7811 7830 1 3 SEQ ID NO: 6117 ggagatgggcggaaacatc 7060 7079 SEQ ID NO: 6663 gatgacccatttcttctcc 8887 8906 1 3 SEQ ID NO: 6118 gagatgggcggaaacatca 7061 7080 SEQ ID NO: 6664 tgatgacccatttcttctc 8886 8905 1 3 SEQ ID NO: 6119 ctagactctttcgagccgc 7112 7131 SEQ ID NO: 6665 gcggcgtgctgacgactag 8457 8476 1 3 SEQ ID NO: 6120 tagactctttcgagccgct 7113 7132 SEQ ID NO: 6666 agcgacgggtcttggtcta 7556 7575 1 3 SEQ ID NO: 6121 agaatgaaatatccattgc 7149 7168 SEQ ID NO: 6667 gcaaagaatgaggttttct 8030 8049 1 3 SEQ ID NO: 6122 ttgcggcggagatcctgcg 7164 7183 SEQ ID NO: 6668 cgcacgatgcatctggcaa 8730 8749 1 3 SEQ ID NO: 6123 agcgaggaggctggtgaga 7580 7599 SEQ ID NO: 6669 tctcgtgcccgaccccgct 9305 9324 1 3 SEQ ID NO: 6124 tgagagcgtcgtctgctgc 7594 7613 SEQ ID NO: 6670 gcagtaaagaccaagctca 9197 9216 1 3 SEQ ID NO: 6125 gtcgtctgctgctcaatgt 7601 7620 SEQ ID NO: 6671 acatggtctacgccacgac 7716 7735 1 3 SEQ ID NO: 6126 tgcgccatcacaacatggt 7704 7723 SEQ ID NO: 6672 accatgtctcccccacgca 6123 6142 1 3 SEQ ID NO: 6127 cagaagaaggtcacctttg 7757 7776 SEQ ID NO: 6673 caaagaatgaggttttctg 8031 8050 1 3 SEQ ID NO: 6128 cctggatgaccattaccgg 7789 7808 SEQ ID NO: 6674 ccggaacctatccagcagg 7936 7955 1 3 SEQ ID NO: 6129 ggacgtgcttaaggagatg 7807 7826 SEQ ID NO: 6675 catcgggccaggagcgtcc 9116 9135 1 3 SEQ ID NO: 6130        aaagaatgaggttttctgc  8032 8051 SEQ ID NO: 6676 gcagaagaaggtcaccttt 7756 7775 1 3 SEQ ID NO: 6131      agttcgtgtatgcgagaag 8110  8129 SEQ ID NO: 6677 cttcatgcctcaggaaact 9069 9088 1 3 SEQ ID NO: 6132       ggctataaaatcgctcaca 8365 8384 SEQ ID NO: 6678 tgtgaaaggtccgtgagcc 9551   9570 1 3 SEQ ID NO: 6133        ttctccatccttctagctc  8900 8919 SEQ ID NO: 6679 gagcggagggggatgagaa 7134 7153 1 3 SEQ ID NO: 6134       tgtctcgtgcccgaccccg  9303 9322 SEQ ID NO: 6680 cggggcgcgttccctgaca 3688 3707 1 3

TABLE 14 Sequences from human hepatitis C virus (HCV) (Direct Match Type) Start End Start End Source Index Index Match Index Index Match # SEQ ID ttttttttttttttttttt 9446 9465 SEQ ID ttttttttttttttttttt 9466 9485 2 NO: 4088 NO: 4661 SEQ ID ttttttttttttttttttt 9446 9465 SEQ ID ttttttttttttttttttt 9465 9484 1 NO: 4347 NO: 5229 SEQ ID ttttttttttttttttttt 9447 9466 SEQ ID ttttttttttttttttttt 9466 9485 1 NO: 4348 NO: 5230

TABLE 15 Sequences of Exemplary Gene Targets gi|4502152|ref|NM_000384.1| Homo sapiens apolipoprotein B (including Ag(x) antigen) (APOB), mRNA ATTCCCACCGGGACCTGCGGGGCTGAGTGCCCTTCTCGGTTGCTGCCGCTGAGGAGCCCGCCCAGCCAGC CAGGGCCGCGAGGCCGAGGCCAGGCCGCAGCCCAGGAGCCGCCCCACCGCAGCTGGCGATGGACCCGCCG AGGCCCGCGCTGCTGGCGCTGCTGGCGCTGCCTGCGCTGCTGCTGCTGCTGCTGGCGGGCGCCAGGGCCG AAGAGGAAATGCTGGAAAATGTCAGCCTGGTCTGTCCAAAAGATGCGACCCGATTCAAGCACCTCCGGAA GTACACATACAACTATGAGGCTGAGAGTTCCAGTGGAGTCCCTGGGACTGCTGATTCAAGAAGTGCCACC AGGATCAACTGCAAGGTTGAGCTGGAGGTTCCCCAGCTCTGCAGCTTCATCCTGAAGACCAGCCAGTGCA CCCTGAAAGAGGTGTATGGCTTCAACCCTGAGGGCAAAGCCTTGCTGAAGAAAACCAAGAACTCTGAGGA GTTTGCTGCAGCCATGTCCAGGTATGAGCTCAAGCTGGCCATTCCAGAAGGGAAGCAGGTTTTCCTTTAC CCGGAGAAAGATGAACCTACTTACATCCTGAACATCAAGAGGGGCATCATTTCTGCCCTCCTGGTTCCCC CAGAGACAGAAGAAGCCAAGCAAGTGTTGTTTCTGGATACCGTGTATGGAAACTGCTCCACTCACTTTAC CGTCAAGACGAGGAAGGGCAATGTGGCAACAGAAATATCCACTGAAAGAGACCTGGGGCAGTGTGATCGC TTCAAGCCCATCCGCACAGGCATCAGCCCACTTGCTCTCATCAAAGGCATGACCCGCCCCTTGTCAACTC TGATCAGCAGCAGCCAGTCCTGTCAGTACACACTGGACGCTAAGAGGAAGCATGTGGCAGAAGCCATCTG CAAGGAGCAACACCTCTTCCTGCCTTTCTCCTACAACAATAAGTATGGGATGGTAGCACAAGTGACACAG ACTTTGAAACTTGAAGACACACCAAAGATCAACAGCCGCTTCTTTGGTGAAGGTACTAAGAAGATGGGCC TCGCATTTGAGAGCACCAAATCCACATCACCTCCAAAGCAGGCCGAAGCTGTTTTGAAGACTCTCCAGGA ACTGAAAAAACTAACCATCTCTGAGCAAAATATCCAGAGAGCTAATCTCTTCAATAAGCTGGTTACTGAG CTGAGAGGCCTCAGTGATGAAGCAGTCACATCTCTCTTGCCACAGCTGATTGAGGTGTCCAGCCCCATCA CTTTACAAGCCTTGGTTCAGTGTGGACAGCCTCAGTGCTCCACTCACATCCTCCAGTGGCTGAAACGTGT GCATGCCAACCCCCTTCTGATAGATGTGGTCACCTACCTGGTGGCCCTGATCCCCGAGCCCTCAGCACAG CAGCTGCGAGAGATCTTCAACATGGCGAGGGATCAGCGCAGCCGAGCCACCTTGTATGCGCTGAGCCACG CGGTCAACAACTATCATAAGACAAACCCTACAGGGACCCAGGAGCTGCTGGACATTGCTAATTACCTGAT GGAACAGATTCAAGATGACTGCACTGGGGATGAAGATTACACCTATTTGATTCTGCGGGTCATTGGAAAT ATGGGCCAAACCATGGAGCAGTTAACTCCAGAACTCAAGTCTTCAATCCTCAAATGTGTCCAAAGTACAA AGCCATCACTGATGATCCAGAAAGCTGCCATCCAGGCTCTGCGGAAAATGGAGCCTAAAGACAAGGACCA GGAGGTTCTTCTTCAGACTTTCCTTGATGATGCTTCTCCGGGAGATAAGCGACTGGCTGCCTATCTTATG TTGATGAGGAGTCCTTCACAGGCAGATATTAACAAAATTGTCCAAATTCTACCATGGGAACAGAATGAGC AAGTGAAGAACTTTGTGGCTTCCCATATTGCCAATATCTTGAACTCAGAAGAATTGGATATCCAAGATCT GAAAAAGTTAGTGAAAGAAGCTCTGAAAGAATCTCAACTTCCAACTGTCATGGACTTCAGAAAATTCTCT CGGAACTATCAACTCTACAAATCTGTTTCTCTTCCATCACTTGACCCAGCCTCAGCCAAAATAGAAGGGA ATCTTATATTTGATCCAAATAACTACCTTCCTAAAGAAAGCATGCTGAAAACTACCCTCACTGCCTTTGG ATTTGCTTCAGCTGACCTCATCGAGATTGGCTTGGAAGGAAAAGGCTTTGAGCCAACATTGGAAGCTCTT TTTGGGAAGCAAGGATTTTTCCCAGACAGTGTCAACAAAGCTTTGTACTGGGTTAATGGTCAAGTTCCTG ATGGTGTCTCTAAGGTCTTAGTGGACCACTTTGGCTATACCAAAGATGATAAACATGAGCAGGATATGGT AAATGGAATAATGCTCAGTGTTGAGAAGCTGATTAAAGATTTGAAATCCAAAGAAGTCCCGGAAGCCAGA GCCTACCTCCGCATCTTGGGAGAGGAGCTTGGTTTTGCCAGTCTCCATGACCTCCAGCTCCTGGGAAAGC TGCTTCTGATGGGTGCCCGCACTCTGCAGGGGATCCCCCAGATGATTGGAGAGGTCATCAGGAAGGGCTC AAAGAATGACTTTTTTCTTCACTACATCTTCATGGAGAATGCCTTTGAACTCCCCACTGGAGCTGGATTA CAGTTGCAAATATCTTCATCTGGAGTCATTGCTCCCGGAGCCAAGGCTGGAGTAAAACTGGAAGTAGCCA ACATGCAGGCTGAACTGGTGGCAAAACCCTCCGTGTCTGTGGAGTTTGTGACAAATATGGGCATCATCAT TCCGGACTTCGCTAGGAGTGGGGTCCAGATGAACACCAACTTCTTCCACGAGTCGGGTCTGGAGGCTCAT GTTGCCCTAAAAGCTGGGAAGCTGAAGTTTATCATTCCTTCCCCAAAGAGACCAGTCAAGCTGCTCAGTG GAGGCAACACATTACATTTGGTCTCTACCACCAAAACGGAGGTGATCCCACCTCTCATTGAGAACAGGCA GTCCTGGTCAGTTTGCAAGCAAGTCTTTCCTGGCCTGAATTACTGCACCTCAGGCGCTTACTCCAACGCC AGCTCCACAGACTCCGCCTCCTACTATCCGCTGACCGGGGACACCAGATTAGAGCTGGAACTGAGGCCTA CAGGAGAGATTGAGCAGTATTCTGTCAGCGCAACCTATGAGCTCCAGAGAGAGGACAGAGCCTTGGTGGA TACCCTGAAGTTTGTAACTCAAGCAGAAGGTGCGAAGCAGACTGAGGCTACCATGACATTCAAATATAAT CGGCAGAGTATGACCTTGTCCAGTGAAGTCCAAATTCCGGATTTTGATGTTGACCTCGGAACAATCCTCA GAGTTAATGATGAATCTACTGAGGGCAAAACGTCTTACAGACTCACCCTGGACATTCAGAACAAGAAAAT TACTGAGGTCGCCCTCATGGGCCACCTAAGTTGTGACACAAAGGAAGAAAGAAAAATCAAGGGTGTTATT TCCATACCCCGTTTGCAAGCAGAAGCCAGAAGTGAGATCCTCGCCCACTGGTCGCCTGCCAAACTGCTTC TCCAAATGGACTCATCTGCTACAGCTTATGGCTCCACAGTTTCCAAGAGGGTGGCATGGCATTATGATGA AGAGAAGATTGAATTTGAATGGAACACAGGCACCAATGTAGATACCAAAAAAATGACTTCCAATTTCCCT GTGGATCTCTCCGATTATCCTAAGAGCTTGCATATGTATGCTAATAGACTCCTGGATCACAGAGTCCCTG AAACAGACATGACTTTCCGGCACGTGGGTTCCAAATTAATAGTTGCAATGAGCTCATGGCTTCAGAAGGC ATCTGGGAGTCTTCCTTATACCCAGACTTTGCAAGACCACCTCAATAGCCTGAAGGAGTTCAACCTCCAG AACATGGGATTGCCAGACTTCCACATCCCAGAAAACCTCTTCTTAAAAAGCGATGGCCGGGTCAAATATA CCTTGAACAAGAACAGTTTGAAAATTGAGATTCCTTTGCCTTTTGGTGGCAAATCCTCCAGAGATCTAAA GATGTTAGAGACTGTTAGGACACCAGCCCTCCACTTCAAGTCTGTGGGATTCCATCTGCCATCTCGAGAG TTCCAAGTCCCTACTTTTACCATTCCCAAGTTGTATCAACTGCAAGTGCCTCTCCTGGGTGTTCTAGACC TCTCCACGAATGTCTACAGCAACTTGTACAACTGGTCCGCCTCCTACAGTGGTGGCAACACCAGCACAGA CCATTTCAGCCTTCGGGCTCGTTACCACATGAAGGCTGACTCTGTGGTTGACCTGCTTTCCTACAATGTG CAAGGATCTGGAGAAACAACATATGACCACAAGAATACGTTCACACTATCATGTGATGGGTCTCTACGCC ACAAATTTCTAGATTCGAATATCAAATTCAGTCATGTAGAAAAACTTGGAAACAACCCAGTCTCAAAAGG TTTACTAATATTCGATGCATCTAGTTCCTGGGGACCACAGATGTCTGCTTCAGTTCATTTGGACTCCAAA AAGAAACAGCATTTGTTTGTCAAAGAAGTCAAGATTGATGGGCAGTTCAGAGTCTCTTCGTTCTATGCTA AAGGCACATATGGCCTGTCTTGTCAGAGGGATCCTAACACTGGCCGGCTCAATGGAGAGTCCAACCTGAG GTTTAACTCCTCCTACCTCCAAGGCACCAACCAGATAACAGGAAGATATGAAGATGGAACCCTCTCCCTC ACCTCCACCTCTGATCTGCAAAGTGGCATCATTAAAAATACTGCTTCCCTAAAGTATGAGAACTACGAGC TGACTTTAAAATCTGACACCAATGGGAAGTATAAGAACTTTGCCACTTCTAACAAGATGGATATGACCTT CTCTAAGCAAAATGCACTGCTGCGTTCTGAATATCAGGCTGATTACGAGTCATTGAGGTTCTTCAGCCTG CTTTCTGGATCACTAAATTCCCATGGTCTTGAGTTAAATGCTGACATCTTAGGCACTGACAAAATTAATA GTGGTGCTCACAAGGCGACACTAAGGATTGGCCAAGATGGAATATCTACCAGTGCAACGACCAACTTGAA GTGTAGTCTCCTGGTGCTGGAGAATGAGCTGAATGCAGAGCTTGGCCTCTCTGGGGCATCTATGAAATTA ACAACAAATGGCCGCTTCAGGGAACACAATGCAAAATTCAGTCTGGATGGGAAAGCCGCCCTCACAGAGC TATCACTGGGAAGTGCTTATCAGGCCATGATTCTGGGTGTCGACAGCAAAAACATTTTCAACTTCAAGGT CAGTCAAGAAGGACTTAAGCTCTCAAATGACATGATGGGCTCATATGCTGAAATGAAATTTGACCACACA AACAGTCTGAACATTGCAGGCTTATCACTGGACTTCTCTTCAAAACTTGACAACATTTACAGCTCTGACA AGTTTTATAAGCAAACTGTTAATTTACAGCTACAGCCCTATTCTCTGGTAACTACTTTAAACAGTGACCT GAAATACAATGCTCTGGATCTCACCAACAATGGGAAACTACGGCTAGAACCCCTGAAGCTGCATGTGGCT GGTAACCTAAAAGGAGCCTACCAAAATAATGAAATAAAACACATCTATGCCATCTCTTCTGCTGCCTTAT CAGCAAGCTATAAAGCAGACACTGTTGCTAAGGTTCAGGGTGTGGAGTTTAGCCATCGGCTCAACACAGA CATCGCTGGGCTGGCTTCAGCCATTGACATGAGCACAAACTATAATTCAGACTCACTGCATTTCAGCAAT GTCTTCCGTTCTGTAATGGCCCCGTTTACCATGACCATCGATGCACATACAAATGGCAATGGGAAACTCG CTCTCTGGGGAGAACATACTGGGCAGCTGTATAGCAAATTCCTGTTGAAAGCAGAACCTCTGGCATTTAC TTTCTCTCATGATTACAAAGGCTCCACAAGTCATCATCTCGTGTCTAGGAAAAGCATCAGTGCAGCTCTT GAACACAAAGTCAGTGCCCTGCTTACTCCAGCTGAGCAGACAGGCACCTGGAAACTCAAGACCCAATTTA ACAACAATGAATACAGCCAGGACTTGGATGCTTACAACACTAAAGATAAAATTGGCGTGGAGCTTACTGG ACGAACTCTGGCTGACCTAACTCTACTAGACTCCCCAATTAAAGTGCCACTTTTACTCAGTGAGCCCATC AATATCATTGATGCTTTAGAGATGAGAGATGCCGTTGAGAAGCCCCAAGAATTTACAATTGTTGCTTTTG TAAAGTATGATAAAAACCAAGATGTTCACTCCATTAACCTCCCATTTTTTGAGACCTTGCAAGAATATTT TGAGAGGAATCGACAAACCATTATAGTTGTAGTGGAAAACGTACAGAGAAACCTGAAGCACATCAATATT GATCAATTTGTAAGAAAATACAGAGCAGCCCTGGGAAAACTCCCACAGCAAGCTAATGATTATCTGAATT CATTCAATTGGGAGAGACAAGTTTCACATGCCAAGGAGAAACTGACTGCTCTCACAAAAAAGTATAGAAT TACAGAAAATGATATACAAATTGCATTAGATGATGCCAAAATCAACTTTAATGAAAAACTATCTCAACTG CAGACATATATGATACAATTTGATCAGTATATTAAAGATAGTTATGATTTACATGATTTGAAAATAGCTA TTGCTAATATTATTGATGAAATCATTGAAAAATTAAAAAGTCTTGATGAGCACTATCATATCCGTGTAAA TTTAGTAAAAACAATCCATGATCTACATTTGTTTATTGAAAATATTGATTTTAACAAAAGTGGAAGTAGT ACTGCATCCTGGATTCAAAATGTGGATACTAAGTACCAAATCAGAATCCAGATACAAGAAAAACTGCAGC AGCTTAAGAGACACATACAGAATATAGACATCCAGCACCTAGCTGGAAAGTTAAAACAACACATTGAGGC TATTGATGTTAGAGTGCTTTTAGATCAATTGGGAACTACAATTTCATTTGAAAGAATAAATGATGTTCTT GAGCATGTCAAACACTTTGTTATAAATCTTATTGGGGATTTTGAAGTAGCTGAGAAAATCAATGCCTTCA GAGCCAAAGTCCATGAGTTAATCGAGAGGTATGAAGTAGACCAACAAATCCAGGTTTTAATGGATAAATT AGTAGAGTTGACCCACCAATACAAGTTGAAGGAGACTATTCAGAAGCTAAGCAATGTCCTACAACAAGTT AAGATAAAAGATTACTTTGAGAAATTGGTTGGATTTATTGATGATGCTGTGAAGAAGCTTAATGAATTAT CTTTTAAAACATTCATTGAAGATGTTAACAAATTCCTTGACATGTTGATAAAGAAATTAAAGTCATTTGA TTACCACCAGTTTGTAGATGAAACCAATGACAAAATCCGTGAGGTGACTCAGAGACTCAATGGTGAAATT CAGGCTCTGGAACTACCACAAAAAGCTGAAGCATTAAAACTGTTTTTAGAGGAAACCAAGGCCACAGTTG CAGTGTATCTGGAAAGCCTACAGGACACCAAAATAACCTTAATCATCAATTGGTTACAGGAGGCTTTAAG TTCAGCATCTTTGGCTCACATGAAGGCCAAATTCCGAGAGACTCTAGAAGATACACGAGACCGAATGTAT CAAATGGACATTCAGCAGGAACTTCAACGATACCTGTCTCTGGTAGGCCAGGTTTATAGCACACTTGTCA CCTACATTTCTGATTGGTGGACTCTTGCTGCTAAGAACCTTACTGACTTTGCAGAGCAATATTCTATCCA AGATTGGGCTAAACGTATGAAAGCATTGGTAGAGCAAGGGTTCACTGTTCCTGAAATCAAGACCATCCTT GGGACCATGCCTGCCTTTGAAGTCAGTCTTCAGGCTCTTCAGAAAGCTACCTTCCAGACACCTGATTTTA TAGTCCCCCTAACAGATTTGAGGATTCCATCAGTTCAGATAAACTTCAAAGACTTAAAAAATATAAAAAT CCCATCCAGGTTTTCCACACCAGAATTTACCATCCTTAACACCTTCCACATTCCTTCCTTTACAATTGAC TTTGTCGAAATGAAAGTAAAGATCATCAGAACCATTGACCAGATGCAGAACAGTGAGCTGCAGTGGCCCG TTCCAGATATATATCTCAGGGATCTGAAGGTGGAGGACATTCCTCTAGCGAGAATCACCCTGCCAGACTT CCGTTTACCAGAAATCGCAATTCCAGAATTCATAATCCCAACTCTCAACCTTAATGATTTTCAAGTTCCT GACCTTCACATACCAGAATTCCAGCTTCCCCACATCTCACACACAATTGAAGTACCTACTTTTGGCAAGC TATACAGTATTCTGAAAATCCAATCTCCTCTTTTCACATTAGATGCAAATGCTGACATAGGGAATGGAAC CACCTCAGCAAACGAAGCAGGTATCGCAGCTTCCATCACTGCCAAAGGAGAGTCCAAATTAGAAGTTCTC AATTTTGATTTTCAAGCAAATGCACAACTCTCAAACCCTAAGATTAATCCGCTGGCTCTGAAGGAGTCAG TGAAGTTCTCCAGCAAGTACCTGAGAACGGAGCATGGGAGTGAAATGCTGTTTTTTGGAAATGCTATTGA GGGAAAATCAAACACAGTGGCAAGTTTACACACAGAAAAAAATACACTGGAGCTTAGTAATGGAGTGATT GTCAAGATAAACAATCAGCTTACCCTGGATAGCAACACTAAATACTTCCACAAATTGAACATCCCCAAAC TGGACTTCTCTAGTCAGGCTGACCTGCGCAACGAGATCAAGACACTGTTGAAAGCTGGCCACATAGCATG GACTTCTTCTGGAAAAGGGTCATGGAAATGGGCCTGCCCCAGATTCTCAGATGAGGGAACACATGAATCA CAAATTAGTTTCACCATAGAAGGACCCCTCACTTCCTTTGGACTGTCCAATAAGATCAATAGCAAACACC TAAGAGTAAACCAAAACTTGGTTTATGAATCTGGCTCCCTCAACTTTTCTAAACTTGAAATTCAATCACA AGTCGATTCCCAGCATGTGGGCCACAGTGTTCTAACTGCTAAAGGCATGGCACTGTTTGGAGAAGGGAAG GCAGAGTTTACTGGGAGGCATGATGCTCATTTAAATGGAAAGGTTATTGGAACTTTGAAAAATTCTCTTT TCTTTTCAGCCCAGCCATTTGAGATCACGGCATCCACAAACAATGAAGGGAATTTGAAAGTTCGTTTTCC ATTAAGGTTAACAGGGAAGATAGACTTCCTGAATAACTATGCACTGTTTCTGAGTCCCAGTGCCCAGCAA GCAAGTTGGCAAGTAAGTGCTAGGTTCAATCAGTATAAGTACAACCAAAATTTCTCTGCTGGAAACAACG AGAACATTATGGAGGCCCATGTAGGAATAAATGGAGAAGCAAATCTGGATTTCTTAAACATTCCTTTAAC AATTCCTGAAATGCGTCTACCTTACACAATAATCACAACTCCTCCACTGAAAGATTTCTCTCTATGGGAA AAAACAGGCTTGAAGGAATTCTTGAAAACGACAAAGCAATCATTTGATTTAAGTGTAAAAGCTCAGTATA AGAAAAACAAACACAGGCATTCCATCACAAATCCTTTGGCTGTGCTTTGTGAGTTTATCAGTCAGAGCAT CAAATCCTTTGACAGGCATTTTGAAAAAAACAGAAACAATGCATTAGATTTTGTCACCAAATCCTATAAT GAAACAAAAATTAAGTTTGATAAGTACAAAGCTGAAAAATCTCACGACGAGCTCCCCAGGACCTTTCAAA TTCCTGGATACACTGTTCCAGTTGTCAATGTTGAAGTGTCTCCATTCACCATAGAGATGTCGGCATTCGG CTATGTGTTCCCAAAAGCAGTCAGCATGCCTAGTTTCTCCATCCTAGGTTCTGACGTCCGTGTGCCTTCA TACACATTAATCCTGCCATCATTAGAGCTGCCAGTCCTTCATGTCCCTAGAAATCTCAAGCTTTCTCTTC CACATTTCAAGGAATTGTGTACCATAAGCCATATTTTTATTCCTGCCATGGGCAATATTACCTATGATTT CTCCTTTAAATCAAGTGTCATCACACTGAATACCAATGCTGAACTTTTTAACCAGTCAGATATTGTTGCT CATCTCCTTTCTTCATCTTCATCTGTCATTGATGCACTGCAGTACAAATTAGAGGGCACCACAAGATTGA CAAGAAAAAGGGGATTGAAGTTAGCCACAGCTCTGTCTCTGAGCAACAAATTTGTGGAGGGTAGTCATAA CAGTACTGTGAGCTTAACCACGAAAAATATGGAAGTGTCAGTGGCAAAAACCACAAAAGCCGAAATTCCA ATTTTGAGAATGAATTTCAAGCAAGAACTTAATGGAAATACCAAGTCAAAACCTACTGTCTCTTCCTCCA TGGAATTTAAGTATGATTTCAATTCTTCAATGCTGTACTCTACCGCTAAAGGAGCAGTTGACCACAAGCT TAGCTTGGAAAGCCTCACCTCTTACTTTTCCATTGAGTCATCTACCAAAGGAGATGTCAAGGGTTCGGTT CTTTCTCGGGAATATTCAGGAACTATTGCTAGTGAGGCCAACACTTACTTGAATTCCAAGAGCACACGGT CTTCAGTGAAGCTGCAGGGCACTTCCAAAATTGATGATATCTGGAACCTTGAAGTAAAAGAAAATTTTGC TGGAGAAGCCACACTCCAACGCATATATTCCCTCTGGGAGCACAGTACGAAAAACCACTTACAGCTAGAG GGCCTCTTTTTCACCAACGGAGAACATACAAGCAAAGCCACCCTGGAACTCTCTCCATGGCAAATGTCAG CTCTTGTTCAGGTCCATGCAAGTCAGCCCAGTTCCTTCCATGATTTCCCTGACCTTGGCCAGGAAGTGGC CCTGAATGCTAACACTAAGAACCAGAAGATCAGATGGAAAAATGAAGTCCGGATTCATTCTGGGTCTTTC CAGAGCCAGGTCGAGCTTTCCAATGACCAAGAAAAGGCACACCTTGACATTGCAGGATCCTTAGAAGGAC ACCTAAGGTTCCTCAAAAATATCATCCTACCAGTCTATGACAAGAGCTTATGGGATTTCCTAAAGCTGGA TGTAACCACCAGCATTGGTAGGAGACAGCATCTTCGTGTTTCAACTGCCTTTGTGTACACCAAAAACCCC AATGGCTATTCATTCTCCATCCCTGTAAAAGTTTTGGCTGATAAATTCATTACTCCTGGGCTGAAACTAA ATGATCTAAATTCAGTTCTTGTCATGCCTACGTTCCATGTCCCATTTACAGATCTTCAGGTTCCATCGTG CAAACTTGACTTCAGAGAAATACAAATCTATAAGAAGCTGAGAACTTCATCATTTGCCCTCAACCTACCA ACACTCCCCGAGGTAAAATTCCCTGAAGTTGATGTGTTAACAAAATATTCTCAACCAGAAGACTCCTTGA TTCCCTTTTTTGAGATAACCGTGCCTGAATCTCAGTTAACTGTGTCCCAGTTCACGCTTCCAAAAAGTGT TTCAGATGGCATTGCTGCTTTGGATCTAAATGCAGTAGCCAACAAGATCGCAGACTTTGAGTTGCCCACC ATCATCGTGCCTGAGCAGACCATTGAGATTCCCTCCATTAAGTTCTCTGTACCTGCTGGAATTGTCATTC CTTCCTTTCAAGCACTGACTGCACGCTTTGAGGTAGACTCTCCCGTGTATAATGCCACTTGGAGTGCCAG TTTGAAAAACAAAGCAGATTATGTTGAAACAGTCCTGGATTCCACATGCAGCTCAACCGTACAGTTCCTA GAATATGAACTAAATGTTTTGGGAACACACAAAATCGAAGATGGTACGTTAGCCTCTAAGACTAAAGGAA CACTTGCACACCGTGACTTCAGTGCAGAATATGAAGAAGATGGCAAATTTGAAGGACTTCAGGAATGGGA AGGAAAAGCGCACCTCAATATCAAAAGCCCAGCGTTCACCGATCTCCATCTGCGCTACCAGAAAGACAAG AAAGGCATCTCCACCTCAGCAGCCTCCCCAGCCGTAGGCACCGTGGGCATGGATATGGATGAAGATGACG ACTTTTCTAAATGGAACTTCTACTACAGCCCTCAGTCCTCTCCAGATAAAAAACTCACCATATTCAAAAC TGAGTTGAGGGTCCGGGAATCTGATGAGGAAACTCAGATCAAAGTTAATTGGGAAGAAGAGGCAGCTTCT GGCTTGCTAACCTCTCTGAAAGACAACGTGCCCAAGGCCACAGGGGTCCTTTATGATTATGTCAACAAGT ACCACTGGGAACACACAGGGCTCACCCTGAGAGAAGTGTCTTCAAAGCTGAGAAGAAATCTGCAGAACAA TGCTGAGTGGGTTTATCAAGGGGCCATTAGGCAAATTGATGATATCGACGTGAGGTTCCAGAAAGCAGCC AGTGGCACCACTGGGACCTACCAAGAGTGGAAGGACAAGGCCCAGAATCTGTACCAGGAACTGTTGACTC AGGAAGGCCAAGCCAGTTTCCAGGGACTCAAGGATAACGTGTTTGATGGCTTGGTACGAGTTACTCAAAA ATTCCATATGAAAGTCAAGCATCTGATTGACTCACTCATTGATTTTCTGAACTTCCCCAGATTCCAGTTT CCGGGGAAACCTGGGATATACACTAGGGAGGAACTTTGCACTATGTTCATAAGGGAGGTAGGGACGGTAC TGTCCCAGGTATATTCGAAAGTCCATAATGGTTCAGAAATACTGTTTTCCTATTTCCAAGACCTAGTGAT TACACTTCCTTTCGAGTTAAGGAAACATAAACTAATAGATGTAATCTCGATGTATAGGGAACTGTTGAAA GATTTATCAAAAGAAGCCCAAGAGGTATTTAAAGCCATTCAGTCTCTCAAGACCACAGAGGTGCTACGTA ATCTTCAGGACCTTTTACAATTCATTTTCCAACTAATAGAAGATAACATTAAACAGCTGAAAGAGATGAA ATTTACTTATCTTATTAATTATATCCAAGATGAGATCAACACAATCTTCAATGATTATATCCCATATGTT TTTAAATTGTTGAAAGAAAACCTATGCCTTAATCTTCATAAGTTCAATGAATTTATTCAAAACGAGCTTC AGGAAGCTTCTCAAGAGTTACAGCAGATCCATCAATACATTATGGCCCTTCGTGAAGAATATTTTGATCC AAGTATAGTTGGCTGGACAGTGAAATATTATGAACTTGAAGAAAAGATAGTCAGTCTGATCAAGAACCTG TTAGTTGCTCTTAAGGACTTCCATTCTGAATATATTGTCAGTGCCTCTAACTTTACTTCCCAACTCTCAA GTCAAGTTGAGCAATTTCTGCACAGAAATATTCAGGAATATCTTAGCATCCTTACCGATCCAGATGGAAA AGGGAAAGAGAAGATTGCAGAGCTTTCTGCCACTGCTCAGGAAATAATTAAAAGCCAGGCCATTGCGACG AAGAAAATAATTTCTGATTACCACCAGCAGTTTAGATATAAACTGCAAGATTTTTCAGACCAACTCTCTG ATTACTATGAAAAATTTATTGCTGAATCCAAAAGATTGATTGACCTGTCCATTCAAAACTACCACACATT TCTGATATACATCACGGAGTTACTGAAAAAGCTGCAATCAACCACAGTCATGAACCCCTACATGAAGCTT GCTCCAGGAGAACTTACTATCATCCTCTAATTTTTTAAAAGAAATCTTCATTTATTCTTCTTTTCCAATT GAACTTTCACATAGCACAGAAAAAATTCAAACTGCCTATATTGATAAAACCATACAGTGAGCCAGCCTTG CAGTAGGCAGTAGACTATAAGCAGAAGCACATATGAACTGGACCTGCACCAAAGCTGGCACCAGGGCTCG GAAGGTCTCTGAACTCAGAAGGATGGCATTTTTTGCAAGTTAAAGAAAATCAGGATCTGAGTTATTTTGC TAAACTTGGGGGAGGAGGAACAAATAAATGGAGTCTTTATTGTGTATCATA (SEQ ID NO: 6681) >gi|4557442|ref|NM_000078.1| Homo sapiens cholesteryl ester transfer protein, plasma (CETP), mRNA GTGAATCTCTGGGGCCAGGAAGACCCTGCTGCCCGGAAGAGCCTCATGTTCCGTGGGGGCTGGGCGGACA TACATATACGGGCTCCAGGCTGAACGGCTCGGGCCACTTACACACCACTGCCTGATAACCATGCTGGCTG CCACAGTCCTGACCCTGGCCCTGCTGGGCAATGCCCATGCCTGCTCCAAAGGCACCTCGCACGAGGCAGG CATCGTGTGCCGCATCACCAAGCCTGCCCTCCTGGTGTTGAACCACGAGACTGCCAAGGTGATCCAGACC GCCTTCCAGCGAGCCAGCTACCCAGATATCACGGGCGAGAAGGCCATGATGCTCCTTGGCCAAGTCAAGT ATGGGTTGCACAACATCCAGATCAGCCACTTGTCCATCGCCAGCAGCCAGGTGGAGCTGGTGGAAGCCAA GTCCATTGATGTCTCCATTCAGAACGTGTCTGTGGTCTTCAAGGGGACCCTGAAGTATGGCTACACCACT GCCTGGTGGCTGGGTATTGATCAGTCCATTGACTTCGAGATCGACTCTGCCATTGACCTCCAGATCAACA CACAGCTGACCTGTGACTCTGGTAGAGTGCGGACCGATGCCCCTGACTGCTACCTGTCTTTCCATAAGCT GCTCCTGCATCTCCAAGGGGAGCGAGAGCCTGGGTGGATCAAGCAGCTGTTCACAAATTTCATCTCCTTC ACCCTGAAGCTGGTCCTGAAGGGACAGATCTGCAAAGAGATCAACGTCATCTCTAACATCATGGCCGATT TTGTCCAGACAAGGGCTGCCAGCATCCTTTCAGATGGAGACATTGGGGTGGACATTTCCCTGACAGGTGA TCCCGTCATCACAGCCTCCTACCTGGAGTCCCATCACAAGGGTCATTTCATCTACAAGAATGTCTCAGAG GACCTCCCCCTCCCCACCTTCTCGCCCACACTGCTGGGGGACTCCCGCATGCTGTACTTCTGGTTCTCTG AGCGAGTCTTCCACTCGCTGGCCAAGGTAGCTTTCCAGGATGGCCGCCTCATGCTCAGCCTGATGGGAGA CGAGTTCAAGGCAGTGCTGGAGACCTGGGGCTTCAACACCAACCAGGAAATCTTCCAAGAGGTTGTCGGC GGCTTCCCCAGCCAGGCCCAAGTCACCGTCCACTGCCTCAAGATGCCCAAGATCTCCTGCCAAAACAAGG GAGTCGTGGTCAATTCTTCAGTGATGGTGAAATTCCTCTTTCCACGCCCAGACCAGCAACATTCTGTAGC TTACACATTTGAAGAGGATATCGTGACTACCGTCCAGGCCTCCTATTCTAAGAAAAAGCTCTTCTTAAGC CTCTTGGATTTCCAGATTACACCAAAGACTGTTTCCAACTTGACTGAGAGCAGCTCCGAGTCCATCCAGA GCTTCCTGCAGTCAATGATCACCGCTGTGGGCATCCCTGAGGTCATGTCTCGGCTCGAGGTAGTGTTTAC AGCCCTCATGAACAGCAAAGGCGTGAGCCTCTTCGACATCATCAACCCTGAGATTATCACTCGAGATGGC TTCCTGCTGCTGCAGATGGACTTTGGCTTCCCTGAGCACCTGCTGGTGGATTTCCTCCAGAGCTTGAGCT AGAAGTCTCCAAGGAGGTCGGGATGGGGCTTGTAGCAGAAGGCAAGCACCAGGCTCACAGCTGGAACCCT GGTGTCTCCTCCAGCGTGGTGGAAGTTGGGTTAGGAGTACGGAGATGGAGATTGGCTCCCAACTCCTCCC TATCCTAAAGGCCCACTGGCATTAAAGTGCTGTATCCAAG (SEQ ID NO: 6682) >gi|414668|emb|X75500.1|HSMTP H. sapiens mRNA for microsomal triglyceride transfer protein TGCAGTTGAGGATTGCTGGTCAATATGATTCTTCTTGCTGTGCTTTTTCTCTGCTTCATTTCCTCATATT CAGCTTCTGTTAAAGGTCACACAACTGGTCTCTCATTAAATAATGACCGGCTGTACAAGCTCACGTACTC CACTGAAGTTCTTCTTGATCGGGGCAAAGGAAAACTGCAAGACAGCGTGGGCTACCGCATTTCCTCCAAC GTGGATGTGGCCTTACTATGGAGGAATCCTGATGGTGATGATGACCAGTTGATCCAAATAACGATGAAGG ATGTAAATGTTGAAAATGTGAATCAGCAGAGAGGAGAGAAGAGCATCTTCAAAGGAAAAAGCCCATCTAA AATAATGGGAAAGGAAAACTTGGAAGCTCTGCAAAGACCTACGCTCCTTCATCTAATCCATGGAAAGGTC AAAGAGTTCTACTCATATCAAAATGAGGCAGTGGCCATAGAAAATATCAAGAGAGGTCTGGCTAGCCTAT TTCAGACACAGTTAAGCTCTGGAACCACCAATGAGGTAGATATCTCTGGAAATTGTAAAGTGACCTACCA GGCTCATCAAGACAAAGTGATCAAAATTAAGGCCTTGGATTCATGCAAAATAGCGAGGTCTGGATTTACG ACCCCAAATCAGGTCTTGGGTGTCAGTTCAAAAGCTACATCTGTCACCACCTATAAGATAGAAGACAGCT TTGTTATAGCTGTGCTTGCTGAAGAAACACACAATTTTGGACTGAATTTCCTACAAACCATTAAGGGGAA AATAGTATCGAAGCAGAAATTAGAGCTGAAGACAACCGAAGCAGGCCCAAGATTGATGTCTGGAAAGCAG GCTGCAGCCATAATCAAAGCAGTTGATTCAAAGTACACGGCCATTCCCATTGTGGGGCAGGTCTTCCAGA GCCACTGTAAAGGATGTCCTTCTCTCTCGGAGCTCTGGCGGTCCACCAGGAAATACCTGCAGCCTGACAA CCTTTCCAAGGCTGAGGCTGTCAGAAACTTCCTGGCCTTCATTCAGCACCTCAGGACTGCGAAGAAAGAA GAGATCCTTCAAATACTAAAGATGGAAAATAAGGAAGTATTACCTCAGCTGGTGGATGCTGTCACCTCTG CTCAGACCTCAGACTCATTAGAAGCCATTTTGGACTTTTTGGATTTCAAAAGTGACAGCAGCATTATCCT CCAGGAGAGGTTTCTCTATGCCTGTGGATTTGCTTCTCATCCCAATGAAGAACTCCTGAGAGCCCTCATT AGTAAGTTCAAAGGTTCTATTGGTAGCAGTGACATCAGAGAAACTGTTATGATCATCACTGGGACACTTG TCAGAAAGTTGTGTCAGAATGAAGGCTGCAAACTCAAAGCAGTAGTGGAAGCTAAGAAGTTAATCCTGGG AGGACTTGAAAAAGCAGAGAAAAAAGAGGACACCAGGATGTATCTGCTGGCTTTGAAGAATGCCCTGCTT CCAGAAGGCATCCCAAGTCTTCTGAAGTATGCAGAAGCAGGAGAAGGGCCCATCAGCCACCTGGCTACCA CTGCTCTCCAGAGATATGATCTCCCTTTCATAACTGATGAGGTGAAGAAGACCTTAAACAGAATATACCA CCAAAACCGTAAAGTTCATGAAAAGACTGTGCGCACTGCTGCAGCTGCTATCATTTTAAATAACAATCCA TCCTACATGGACGTCAAGAACATCCTGCTGTCTATTGGGGAGCTTCCCCAAGAAATGAATAAATACATGC TCGCCATTGTTCAAGACATCCTACGTTTTGAAATGCCTGCAAGCAAAATTGTCCGTCGAGTTCTGAAGGA AATGGTCGCTCACAATTATGACCGTTTCTCCAGGAGTGGATCTTCTTCTGCCTACACTGGCTACATAGAA CGTAGTCCCCGTTCGGCATCTACTTACAGCCTAGACATTCTCTACTCGGGTTCTGGCATTCTAAGGAGAA GTAACCTGAACATCTTTCAGTACATTGGGAAGGCTGGTCTTCACGGTAGCCAGGTGGTTATTGAAGCCCA AGGACTGGAAGCCTTAATCGCAGCCACCCCTGACGAGGGGGAGGAGAACCTTGACTCCTATGCTGGTATG TCAGCCATCCTCTTTGATGTTCAGCTCAGACCTGTCACCTTTTTCAACGGATACAGTGATTTGATGTCCA AAATGCTGTCAGCATCTGGCGACCCTATCAGTGTGGTGAAAGGACTTATTCTGCTAATAGATCATTCTCA GGAACTTCAGTTACAATCTGGACTAAAAGCCAATATAGAGGTCCAGGGTGGTCTAGCTATTGATATTTCA GGTGCAATGGAGTTTAGCTTGTGGTATCGTGAGTCTAAAACCCGAGTGAAAAATAGGGTGACTGTGGTAA TAACCACTGACATCACAGTGGACTCCTCTTTTGTGAAAGCTGGCCTGGAAACCAGTACAGAAACAGAAGC AGGCTTGGAGTTTATCTCCACAGTGCAGTTTTCTCAGTACCCATTCTTAGTTTGCATGCAGATGGACAAG GATGAAGCTCCATTCAGGCAATTTGAGAAAAAGTACGAAAGGCTGTCCACAGGCAGAGGTTATGTCTCTC AGAAAAGAAAAGAAAGCGTATTAGCAGGATGTGAATTCCCGCTCCATCAAGAGAACTCAGAGATGTGCAA AGTGGTGTTTGCCCCTCAGCCGGATAGTACTTCCAGCGGATGGTTTTGAAACTGACCTGTGATATTTTAC TTGAATTTGTCTCCCCGAAAGGGACACAATGTGGCATGACTAAGTACTTGCTCTCTGAGAGCACAGCGTT TACATATTTACCTGTATTTAAGATTTTTGTAAAAAGCTACAAAAAACTGCAGTTTGATCAAATTTGGGTA TATGCAGTATGCTACCCACAGCGTCATTTTGAATCATCATGTGACGCTTTCAACAACGTTCTTAGTTTAC TTATACCTCTCTCAAATCTCATTTGGTACAGTCAGAATAGTTATTCTCTAAGAGGAAACTAGTGTTTGTT AAAAACAAAAATAAAAACAAAACCACACAAGGAGAACCCAATTTTGTTTCAACAATTTTTGATCAATGTA TATGAAGCTCTTGATAGGACTTCCTTAAGCATGACGGGAAAACCAAACACGTTCCCTAATCAGGAAAAAA AAAAAAAAAAAAAAGTAAGACACAAACAAACCATTTTTTTCTCTTTTTTTGGAGTTGGGGGCCCAGGGAG AAGGGACAAGGCTTTTAAAAGACTTGTTAGCCAACTTCAAGAATTAATATTTATGTCTCTGTTATTGTTA GTTTTAAGCCTTAAGGTAGAAGGCACATAGAAATAACATC (SEQ ID NO: 6683) >gi|1217638|emb|X91148.1|HSMTTP H. sapiens mRNA for microsomal triglyceride transfer protein TGCAGTTGAGGATTGCTGGTCAATATGATTCTTCTTGCTGTGCTTTTTCTCTGCTTCATTTCCTCATATT CAGCTTCTGTTAAAGGTCACACAACTGGTCTCTCATTAAATAATGACCGGCTGTACAAGCTCACGTACTC CACTGAAGTTCTTCTTGATCGGGGCAAAGGAAAACTGCAAGACAGCGTGGGCTACCGCATTTCCTCCAAC GTGGATGTGGCCTTACTATGGAGGAATCCTGATGGTGATGATGACCAGTTGATCCAAATAACGATGAAGG ATGTAAATGTTGAAAATGTGAATCAGCAGAGAGGAGAGAAGAGCATCTTCAAAGGAAAAAGCCCATCTAA AATAATGGGAAAGGAAAACTTGGAAGCTCTGCAAAGACCTACGCTCCTTCATCTAATCCATGGAAAGGTC AAAGAGTTCTACTCATATCAAAATGAGGCAGTGGCCATAGAAAATATCAAGAGAGGTCTGGCTAGCCTAT TTCAGACACAGTTAAGCTCTGGAACCACCAATGAGGTAGATATCTCTGGAAATTGTAAAGTGACCTACCA GGCTCATCAAGACAAAGTGATCAAAATTAAGGCCTTGGATTCATGCAAAATAGCGAGGTCTGGATTTACG ACCCCAAATCAGGTCTTGGGTGTCAGTTCAAAAGCTACATCTGTCACCACCTATAAGATAGAAGACAGCT TTGTTATAGCTGTGCTTGCTGAAGAAACACACAATTTTGGACTGAATTTCCTACAAACCATTAAGGGGAA AATAGTATCGAAGCAGAAATTAGAGCTGAAGACAACCGAAGCAGGCCCAAGATTGATGTCTGGAAAGCAG GCTGCAGCCATAATCAAAGCAGTTGATTCAAAGTACACGGCCATTCCCATTGTGGGGCAGGTCTTCCAGA GCCACTGTAAAGGATGTCCTTCTCTCTCGGAGCTCTGGCGGTCCACCAGGAAATACCTGCAGCCTGACAA CCTTTCCAAGGCTGAGGCTGTCAGAAACTTCCTGGCCTTCATTCAGCACCTCAGGACTGCGAAGAAAGAA GAGATCCTTCAAATACTAAAGATGGAAAATAAGGAAGTATTACCTCAGCTGGTGGATGCTGTCACCTCTG CTCAGACCTCAGACTCATTAGAAGCCATTTTGGACTTTTTGGATTTCAAAAGTGACAGCAGCATTATCCT CCAGGAGAGGTTTCTCTATGCCTGTGGATTTGCTTCTCATCCCAATGAAGAACTCCTGAGAGCCCTCATT AGTAAGTTCAAAGGTTCTATTGGTAGCAGTGACATCAGAGAAACTGTTATGATCATCACTGGGACACTTG TCAGAAAGTTGTGTCAGAATGAAGGCTGCAAACTCAAAGCAGTAGTGGAAGCTAAGAAGTTAATCCTGGG AGGACTTGAAAAAGCAGAGAAAAAAGAGGACACCAGGATGTATCTGCTGGCTTTGAAGAATGCCCTGCTT CCAGAAGGCATCCCAAGTCTTCTGAAGTATGCAGAAGCAGGAGAAGGGCCCATCAGCCACCTGGCTACCA CTGCTCTCCAGAGATATGATGCTCCCTTTCATAACTGATGAGGTGAAGAAGACCTTAAACAGAATATACC ACCAAAACCGTAAAGTTCATGAAAAGACTGTGCGCACTGCTGCAGCTGCTATCATTTTAAATAACAATCC ATCCTACATGGACGTCAAGAACATCCTGCTGTCTATTGGGGAGCTTCCCCAAGAAATGAATAAATACATG CTCGCCATTGTTCAAGACATCCTACGTTTTGAAATGCCTGCAAGCAAAATTGTCCGTCGAGTTCTGAAGG AAATGGTCGCTCACAATTATGACCGTTTCTCCAGGAGTGGATCTTCTTCTGCCTACACTGGCTACATAGA ACGTAGTCCCCGTTCGGCATCTACTTACAGCCTAGACATTCTCTACTCGGGTTCTGGCATTCTAAGGAGA AGTAACCTGAACATCTTTCAGTACATTGGGAAGGCTGGTCTTCACGGTAGCCAGGTGGTTATTGAAGCCC AAGGACTGGAAGCCTTAATCGCAGCCACCCCTGACGAGGGGGAGGAGAACCTTGACTCCTATGCTGGTAT GTCAGCCATCCTCTTTGATGTTCAGCTCAGACCTGTCACCTTTTTCAACGGATACAGTGATTTGATGTCC AAAATGCTGTCAGCATCTGGCGACCCTATCAGTGTGGTGAAAGGACTTATTCTGCTAATAGATCATTCTC AGGAACTTCAGTTACAATCTGGACTAAAAGCCAATATAGAGGTCCAGGGTGGTCTAGCTATTGATATTTC AGGTGCAATGGAGTTTAGCTTGTGGTATCGTGAGTCTAAAACCCGAGTGAAAAATAGGGTGACTGTGGTA ATAACCACTGACATCACAGTGGACTCCTCTTTTGTGAAAGCTGGCCTGGAAACCAGTACAGAAACAGAAG CAGGCTTGGAGTTTATCTCCACAGTGCAGTTTTCTCAGTACCCATTCTTAGTTTGCATGCAGATGGACAA GGATGAAGCTCCATTCAGGCAATTTGAGAAAAAGTACGAAAGGCTGTCCACAGGCAGAGGTTATGTCTCT CAGAAAAGAAAAGAAAGCGTATTAGCAGGATGTGAATTCCCGCTCCATCAAGAGAACTCAGAGATGTGCA AAGTGGTGTTTGCCCCTCAGCCGGATAGTACTTCCAGCGGATGGTTTTGAAACTGACCTGTGATATTTTA CTTGAATTTGTCTCCCCGAAAGGGACACAATGTGGCATGACTAAGTACTTGCTCTCTGAGAGCACAGCGT TTACATATTTACCTGTATTTAAGATTTTTGTAAAAAGCTACAAAAAACTGCAGTTTGATCAAATTTGGGT ATATGCAGTATGCTACCCACAGCGTCATTTTGAATCATCATGTGACGCTTTCAACAACGTTCTTAGTTTA CTTATACCTCTCTCAAATCTCATTTGGTACAGTCAGAATAGTTATTCTCTAAGAGGAAACTAGTGTTTGT TAAAAACAAAAATAAAAACAAAACCACACAAGGAGAACCCAATTTTGTTTCAACAATTTTTGATCAATGT ATATGAAGCTCTTGATAGGACTTCCTTAAGCATGACGGGAAAACCAAACACGTTCCCTAATCAGGAAAAA AAAAAAAAAAGAAAAAGTAAGACACAAACAAACCATTTTTTTCTCTTTTTTTGGAGTTGGGGGCCCAGGG AGAAGGGACAAGGCTTTTAAAAGACTTGTTAGCCAACTTCAAGAATTAATATTTATGTCTCTGTTATTGT TAGTTTTAAGCCTTAAGGTAGAAGGCACATAGAAATAACATCTCATCTTTCTGCTGACCATTTTAGTGAG GTTGTTCCAAAGAGCATTCAGGTCTCTACCTCCAGCCCTGCAAAAATATTGGACCTAGCACAGAGGAATC AGGAAAATTAATTTCAGAAACTCCATTTGATTTTTCTTTTGCTGTGTCTTTTTTGAGACTGTAATATGGT ACACTGTCCTCTAAGGACATCCTCATTTTATCTCACCTTTTTGGGGGTGAGAGCTCTAGTTCATTTAACT GTACTCTGCACAATAGCTAGGATGACTAAGAGAACATTGCTTCAAGAAACTGGTGGATTTGGATTTCCAA AATATGAAATAAGGAGAAAAATGTTTTTATTTGTATGAATTAAAAGATCCATGTTGAACATTTGCAAATA TTTATTAATAAACAGATGTGGTGATAAACCCAAAACAAATGACAGGTGCTTATTTTCCACTAAACACAGA CACATGAAATGAAAGTTTAGCTAGCCCACTATTTGTTGTAAATTGAAAACGAAGTGTGATAAAATAAATA TGTAGAAATCAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 6684) >gi|21361125|ref|NM_001467.2| Homo sapiens glucose-6-phosphatase, transport (glucose-6-phosphate) protein 1 (G6PT1), mRNA GGCACGAGGGGCCACCGAGGCGCTGTCCCTGACCACCAGCACGAGACCCCTTTCTATCGCGCCAGTCCTG TGGTCTCCGCACCTCTCCAGCTCCTGCACCCCCGGCCCCCGTGGTTCCCAGCCGCACAGTAGCGTGTCCT GGGTAGCGTGAGGACCCACGGGGCTGAGCAGGTGCCACGAGCCCGCCGCCTCTTCGCCGCCCGCCGCCTC TCCTCCTCTCCCGCCCGCCGCCTGGCCCTCCCCTACCAGGCTGAGCCTCTGGCTGCCAGAAGCGCGGGGC CTCCGGGAGAATACGTGCGGTCGCCCGCTCCGCGTGCGCCTACGCCTTCTGCTCCAGTTGCTTTCCCAAT TGAGCGGAAAAGCCGGGGCATGTTGCCGGGGCCCTGGGCGGGACGGTTGTGCCCTGCAGCCCGAAGCCCG CCGGGGCACCTTCCCGCCCACGAGCTGCCCAGTCCCTCTGCTTGCGGCCCCTGCCAACGTCCCACAGGAC ACTGGGTCCCCTTGGAGCCTCCCCAGGCTTAATGATTGTCCAGAAGGCGGCTATAAAGGGAGCCTGGGAG GCTGGGTGGAGGAGGGAGCAGAAAAAACCCAACTCAGCAGATCTGGGAACTGTGAGAGCGGCAAGCAGGA ACTGTGGTCAGAGGCTGTGCGTCTTGGCTGGTAGGGCCTGCTCTTTTCTACCATGGCAGCCCAGGGCTAT GGCTATTATCGCACTGTGATCTTCTCAGCCATGTTTGGGGGCTACAGCCTGTATTACTTCAATCGCAAGA CCTTCTCCTTTGTCATGCCATCATTGGTGGAAGAGATCCCTTTGGACAAGGATGATTTGGGGTTCATCAC CAGCAGCCAGTCGGCAGCTTATGCTATCAGCAAGTTTGTCAGTGGGGTGCTGTCTGACCAGATGAGTGCT CGCTGGCTCTTCTCTTCTGGGCTGCTCCTGGTTGGCCTGGTCAACATATTCTTTGCCTGGAGCTCCACAG TACCTGTCTTTGCTGCCCTCTGGTTCCTTAATGGCCTGGCCCAGGGGCTGGGCTGGCCCCCATGTGGGAA GGTCCTGCGGAAGTGGTTTGAGCCATCTCAGTTTGGCACTTGGTGGGCCATCCTGTCAACCAGCATGAAC CTGGCTGGAGGGCTGGGCCCTATCCTGGCAACCATCCTTGCCCAGAGCTACAGCTGGCGCAGCACGCTGG CCCTATCTGGGGCACTGTGTGTGGTTGTCTCCTTCCTCTGTCTCCTGCTCATCCACAATGAACCTGCTGA TGTTGGACTCCGCAACCTGGACCCCATGCCCTCTGAGGGCAAGAAGGGCTCCTTGAAGGAGGAGAGCACC CTGCAGGAGCTGCTGCTGTCCCCTTACCTGTGGGTGCTCTCCACTGGTTACCTTGTGGTGTTTGGAGTAA AGACCTGCTGTACTGACTGGGGCCAGTTCTTCCTTATCCAGGAGAAAGGACAGTCAGCCCTTGTAGGTAG CTCCTACATGAGTGCCCTGGAAGTTGGGGGCCTTGTAGGCAGCATCGCAGCTGGCTACCTGTCAGACCGG GCCATGGCAAAGGCGGGACTGTCCAACTACGGGAACCCTCGCCATGGCCTGTTGCTGTTCATGATGGCTG GCATGACAGTGTCCATGTACCTCTTCCGGGTAACAGTGACCAGTGACTCCCCCAAGCTCTGGATCCTGGT ATTGGGAGCTGTATTTGGTTTCTCCTCGTATGGCCCCATTGCCCTGTTTGGAGTCATAGCCAACGAGAGT GCCCCTCCCAACTTGTGTGGCACCTCCCACGCCATTGTGGGACTCATGGCCAATGTGGGCGGCTTTCTGG CTGGGCTGCCCTTCAGCACCATTGCCAAGCACTACAGTTGGAGCACAGCCTTCTGGGTGGCTGAAGTGAT TTGTGCGGCCAGCACGGCTGCCTTCTTCCTCCTACGAAACATCCGCACCAAGATGGGCCGAGTGTCCAAG AAGGCTGAGTGAAGAGAGTCCAGGTTCCGGAGCACCATCCCACGGTGGCCTTCCCCCTGCACGCTCTGCG GGGAGAAAAGGAGGGGCCTGCCTGGCTAGCCCTGAACCTTTCACTTTCCATTTCTGCGCCTTTTCTGTCA CCCGGGTGGCGCTGGAAGTTATCAGTGGCTAGTGAGGTCCCAGCTCCCTGATCCTATGCTCTATTTAAAA GATAACCTTTGGCCTTAGACTCCGTTAGCTCCTATTTCCTGCCTTCAGACAAACAGGAAACTTCTGCAGT CAGGAAGGCTCCTGTACCCTTCTTCTTTTCCTAGGCCCTGTCCTGCCCGCATCCTACCCCATCCCCACCT GAAGTGAGGCTATCCCTGCAGCTGCAGGGCACTAATGACCCTTGACTTCTGCTGGGTCCTAAGTCCTCTC AGCAGTGGGTGACTGCTGTTGCCAATACCTCAGACTCCAGGGAAAGAGAGGAGGCCATCATTCTCACTGT ACCACTAGGCGCAGTTGGATATAGGTGGGAAGAAAAGGTGACTTGTTATAGAAGATTAAAACTAGATTTG ATACTGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 6685) gi|4503130|ref|NM_001904.1| Homo sapiens catenin (cadherin-associated protein), beta 1, 88 kDa (CTNNB1), mRNA AAGCCTCTCGGTCTGTGGCAGCAGCGTTGGCCCGGCCCCGGGAGCGGAGAGCGAGGGGAGGCGGAGACGG AGGAAGGTCTGAGGAGCAGCTTCAGTCCCCGCCGAGCCGCCACCGCAGGTCGAGGACGGTCGGACTCCCG CGGCGGGAGGAGCCTGTTCCCCTGAGGGTATTTGAAGTATACCATACAACTGTTTTGAAAATCCAGCGTG GACAATGGCTACTCAAGCTGATTTGATGGAGTTGGACATGGCCATGGAACCAGACAGAAAAGCGGCTGTT AGTCACTGGCAGCAACAGTCTTACCTGGACTCTGGAATCCATTCTGGTGCCACTACCACAGCTCCTTCTC TGAGTGGTAAAGGCAATCCTGAGGAAGAGGATGTGGATACCTCCCAAGTCCTGTATGAGTGGGAACAGGG ATTTTCTCAGTCCTTCACTCAAGAACAAGTAGCTGATATTGATGGACAGTATGCAATGACTCGAGCTCAG AGGGTACGAGCTGCTATGTTCCCTGAGACATTAGATGAGGGCATGCAGATCCCATCTACACAGTTTGATG CTGCTCATCCCACTAATGTCCAGCGTTTGGCTGAACCATCACAGATGCTGAAACATGCAGTTGTAAACTT GATTAACTATCAAGATGATGCAGAACTTGCCACACGTGCAATCCCTGAACTGACAAAACTGCTAAATGAC GAGGACCAGGTGGTGGTTAATAAGGCTGCAGTTATGGTCCATCAGCTTTCTAAAAAGGAAGCTTCCAGAC ACGCTATCATGCGTTCTCCTCAGATGGTGTCTGCTATTGTACGTACCATGCAGAATACAAATGATGTAGA AACAGCTCGTTGTACCGCTGGGACCTTGCATAACCTTTCCCATCATCGTGAGGGCTTACTGGCCATCTTT AAGTCTGGAGGCATTCCTGCCCTGGTGAAAATGCTTGGTTCACCAGTGGATTCTGTGTTGTTTTATGCCA TTACAACTCTCCACAACCTTTTATTACATCAAGAAGGAGCTAAAATGGCAGTGCGTTTAGCTCGTGGGCT GCAGAAAATGGTTGCCTTGCTCAACAAAACAAATGTTAAATTCTTGGCTATTACGACAGACTGCCTTCAA ATTTTAGCTTATGGCAACCAAGAAAGCAAGCTCATCATACTGGCTAGTGGTGGACCCCAAGCTTTAGTAA ATATAATGAGGACCTATACTTACGAAAAACTACTGTGGACCACAAGCAGAGTGCTGAAGGTGCTATCTGT CTGCTCTAGTAATAAGCCGGCTATTGTAGAAGCTGGTGGAATGCAAGCTTTAGGACTTCACCTGACAGAT CCAAGTCAACGTCTTGTTCAGAACTGTCTTTGGACTCTCAGGAATCTTTCAGATGCTGCAACTAAACAGG AAGGGATGGAAGGTCTCCTTGGGACTCTTGTTCAGCTTCTGGGTTCAGATGATATAAATGTGGTCACCTG TGCAGCTGGAATTCTTTCTAACCTCACTTGCAATAATTATAAGAACAAGATGATGGTCTGCCAAGTGGGT GGTATAGAGGCTCTTGTGCGTACTGTCGTTCGGGCTGGTGACAGGGAAGACATCACTGAGCCTGCCATCT GTGCTCTTCGTCATCTGACCAGCCGACACCAAGAAGCAGAGATGGCCCAGAATGCAGTTCGCCTTCACTA TGGACTACCAGTTGTGGTTAAGCTCTTACACCCACCATCCCACTGGCCTCTGATAAAGGCTACTGTTGGA TTGATTCGAAATCTTGCCCTTTGTCCCGCAAATCATGCACCTTTGCGTGAGCAGGGTGCCATTCCACGAC TAGTTCAGTTGCTTGTTCGTGCACATCAGGATACCCAGCGCCGTACGTCCATGGGTGGGACACAGCAGCA ATTTGTGGAGGGGGTCCGCATGGAAGAAATAGTTGAAGGTTGTACCGGAGCCCTTCACATCCTAGCTCGG GATGTTCACAACCGAATTGTTATCAGAGGACTAAATACCATTCCATTGTTTGTGCAGCTGCTTTATTCTC CCATTGAAAACATCCAAAGAGTAGCTGCAGGGGTCCTCTGTGAACTTGCTCAGGACAAGGAAGCTGCAGA AGCTATTGAAGCTGAGGGAGCCAGAGCTCCTCTGACAGAGTTACTTCACTCTAGGAATGAAGGTGTGGCG ACATATGCAGCTGCTGTTTTGTTCCGAATGTCTGAGGACAAGCCACAAGATTACAAGAAACGGCTTTCAG TTGAGCTGACCAGCTCTCTCTTCAGAACAGAGCCAATGGCTTGCAATGAGACTGCTGATCTTGGACTTGA TATTGGTGCCCAGGGAGAACCCCTTGGATATCGCCAGGATGATCCTAGCTATCGTTCTTTTCACTCTGGT GGATATGGGCAGGATGCCTTGGGTATGGACCCCATGATGGAACATGAGATGGGTGGCCACCACCCTGGTG CTGACTATCCAGTTGATGGGCTGCCAGATCTGGGGCATGCCCAGGACCTCATGGATGGGCTGCCTCCAGG TGACAGCAATCAGCTGGCCTGGTTTGATACTGACCTGTAAATCATCCTTTAGCTGTATTGTCTGAACTTG CATTGTGATTGGCCTGTAGAGTTGCTGAGAGGGCTCGAGGGGTGGGCTGGTATCTCAGAAAGTGCCTGAC ACACTAACCAAGCTGAGTTTCCTATGGGAACAATTGAAGTAAACTTTTTGTTCTGGTCCTTTTTGGTCGA GGAGTAACAATACAAATGGATTTTGGGAGTGACTCAAGAAGTGAAGAATGCACAAGAATGGATCACAAGA TGGAATTTAGCAAACCCTAGCCTTGCTTGTTAAAATTTTTTTTTTTTTTTTTTTAAGAATATCTGTAATG GTACTGACTTTGCTTGCTTTGAAGTAGCTCTTTTTTTTTTTTTTTTTTTTTTTTTTTGCAGTAACTGTTT TTTAAGTCTCTCGTAGTGTTAAGTTATAGTGAATACTGCTACAGCAATTTCTAATTTTTAAGAATTGAGT AATGGTGTAGAACACTAATTAATTCATAATCACTCTAATTAATTGTAATCTGAATAAAGTGTAACAATTG TGTAGCCTTTTTGTATAAAATAGACAAATAGAAAATGGTCCAATTAGTTTCCTTTTTAATATGCTTAAAA TAAGCAGGTGGATCTATTTCATGTTTTTGATCAAAAACTATTTGGGATATGTATGGGTAGGGTAAATCAG TAAGAGGTGTTATTTGGAACCTTGTTTTGGACAGTTTACCAGTTGCCTTTTATCCCAAAGTTGTTGTAAC CTGCTGTGATACGATGCTTCAAGAGAAAATGCGGTTATAAAAAATGGTTCAGAATTAAACTTTTAATTCA TT (SEQ ID NO: 6686) gi|18104977|ref|NM_002827.2| Homo sapiens protein tyrosine phosphatase, non-receptor type 1 (PTPN1), mRNA GTGATGCGTAGTTCCGGCTGCCGGTTGACATGAAGAAGCAGCAGCGGCTAGGGCGGCGGTAGCTGCAGGG GTCGGGGATTGCAGCGGGCCTCGGGGCTAAGAGCGCGACGCGGCCTAGAGCGGCAGACGGCGCAGTGGGC CGAGAAGGAGGCGCAGCAGCCGCCCTGGCCCGTCATGGAGATGGAAAAGGAGTTCGAGCAGATCGACAAG TCCGGGAGCTGGGCGGCCATTTACCAGGATATCCGACATGAAGCCAGTGACTTCCCATGTAGAGTGGCCA AGCTTCCTAAGAACAAAAACCGAAATAGGTACAGAGACGTCAGTCCCTTTGACCATAGTCGGATTAAACT ACATCAAGAAGATAATGACTATATCAACGCTAGTTTGATAAAAATGGAAGAAGCCCAAAGGAGTTACATT CTTACCCAGGGCCCTTTGCCTAACACATGCGGTCACTTTTGGGAGATGGTGTGGGAGCAGAAAAGCAGGG GTGTCGTCATGCTCAACAGAGTGATGGAGAAAGGTTCGTTAAAATGCGCACAATACTGGCCACAAAAAGA AGAAAAAGAGATGATCTTTGAAGACACAAATTTGAAATTAACATTGATCTCTGAAGATATCAAGTCATAT TATACAGTGCGACAGCTAGAATTGGAAAACCTTACAACCCAAGAAACTCGAGAGATCTTACATTTCCACT ATACCACATGGCCTGACTTTGGAGTCCCTGAATCACCAGCCTCATTCTTGAACTTTCTTTTCAAAGTCCG AGAGTCAGGGTCACTCAGCCCGGAGCACGGGCCCGTTGTGGTGCACTGCAGTGCAGGCATCGGCAGGTCT GGAACCTTCTGTCTGGCTGATACCTGCCTCTTGCTGATGGACAAGAGGAAAGACCCTTCTTCCGTTGATA TCAAGAAAGTGCTGTTAGAAATGAGGAAGTTTCGGATGGGGCTGATCCAGACAGCCGACCAGCTGCGCTT CTCCTACCTGGCTGTGATCGAAGGTGCCAAATTCATCATGGGGGACTCTTCCGTGCAGGATCAGTGGAAG GAGCTTTCCCACGAGGACCTGGAGCCCCCACCCGAGCATATCCCCCCACCTCCCCGGCCACCCAAACGAA TCCTGGAGCCACACAATGGGAAATGCAGGGAGTTCTTCCCAAATCACCAGTGGGTGAAGGAAGAGACCCA GGAGGATAAAGACTGCCCCATCAAGGAAGAAAAAGGAAGCCCCTTAAATGCCGCACCCTACGGCATCGAA AGCATGAGTCAAGACACTGAAGTTAGAAGTCGGGTCGTGGGGGGAAGTCTTCGAGGTGCCCAGGCTGCCT CCCCAGCCAAAGGGGAGCCGTCACTGCCCGAGAAGGACGAGGACCATGCACTGAGTTACTGGAAGCCCTT CCTGGTCAACATGTGCGTGGCTACGGTCCTCACGGCCGGCGCTTACCTCTGCTACAGGTTCCTGTTCAAC AGCAACACATAGCCTGACCCTCCTCCACTCCACCTCCACCCACTGTCCGCCTCTGCCCGCAGAGCCCACG CCCGACTAGCAGGCATGCCGCGGTAGGTAAGGGCCGCCGGACCGCGTAGAGAGCCGGGCCCCGGACGGAC GTTGGTTCTGCACTAAAACCCATCTTCCCCGGATGTGTGTCTCACCCCTCATCCTTTTACTTTTTGCCCC TTCCACTTTGAGTACCAAATCCACAAGCCATTTTTTGAGGAGAGTGAAAGAGAGTACCATGCTGGCGGCG CAGAGGGAAGGGGCCTACACCCGTCTTGGGGCTCGCCCCACCCAGGGCTCCCTCCTGGAGCATCCCAGGC GGGCGGCACGCCAACAGCCCCCCCCTTGAATCTGCAGGGAGCAACTCTCCACTCCATATTTATTTAAACA ATTTTTTCCCCAAAGGCATCCATAGTGCACTAGCATTTTCTTGAACCAATAATGTATTAAAATTTTTTGA TGTCAGCCTTGCATCAAGGGCTTTATCAAAAAGTACAATAATAAATCCTCAGGTAGTACTGGGAATGGAA GGCTTTGCCATGGGCCTGCTGCGTCAGACCAGTACTGGGAAGGAGGACGGTTGTAAGCAGTTGTTATTTA GTGATATTGTGGGTAACGTGAGAAGATAGAACAATGCTATAATATATAATGAACACGTGGGTATTTAATA AGAAACATGATGTGAGATTACTTTGTCCCGCTTATTCTCCTCCCTGTTATCTGCTAGATCTAGTTCTCAA TCACTGCTCCCCCGTGTGTATTAGAATGCATGTAAGGTCTTCTTGTGTCCTGATGAAAAATATGTGCTTG AAATGAGAAACTTTGATCTCTGCTTACTAATGTGCCCCATGTCCAAGTCCAACCTGCCTGTGCATGACCT GATCATTACATGGCTGTGGTTCCTAAGCCTGTTGCTGAAGTCATTGTCGCTCAGCAATAGGGTGCAGTTT TCCAGGAATAGGCATTTGCCTAATTCCTGGCATGACACTCTAGTGACTTCCTGGTGAGGCCCAGCCTGTC CTGGTACAGCAGGGTCTTGCTGTAACTCAGACATTCCAAGGGTATGGGAAGCCATATTCACACCTCACGC TCTGGACATGATTTAGGGAAGCAGGGACACCCCCCGCCCCCCACCTTTGGGATCAGCCTCCGCCATTCCA AGTCAACACTCTTCTTGAGCAGACCGTGATTTGGAAGAGAGGCACCTGCTGGAAACCACACTTCTTGAAA CAGCCTGGGTGACGGTCCTTTAGGCAGCCTGCCGCCGTCTCTGTCCCGGTTCACCTTGCCGAGAGAGGCG CGTCTGCCCCACCCTCAAACCCTGTGGGGCCTGATGGTGCTCACGACTCTTCCTGCAAAGGGAACTGAAG ACCTCCACATTAAGTGGCTTTTTAACATGAAAAACACGGCAGCTGTAGCTCCCGAGCTACTCTCTTGCCA GCATTTTCACATTTTGCCTTTCTCGTGGTAGAAGCCAGTACAGAGAAATTCTGTGGTGGGAACATTCGAG GTGTCACCCTGCAGAGCTATGGTGAGGTGTGGATAAGGCTTAGGTGCCAGGCTGTAAGCATTCTGAGCTG GGCTTGTTGTTTTTAAGTCCTGTATATGTATGTAGTAGTTTGGGTGTGTATATATAGTAGCATTTCAAAA TGGACGTACTGGTTTAACCTCCTATCCTTGGAGAGCAGCTGGCTCTCCACCTTGTTACACATTATGTTAG AGAGGTAGCGAGCTGCTCTGCTATATGCCTTAAGCCAATATTTACTCATCAGGTCATTATTTTTTACAAT GGCCATGGAATAAACCATTTTTACAAAA (SEQ ID NO: 6687) gi|12831192|gb|AF333324.1|Hepatitis C virus type 1b polyprotein mRNA, complete cds GCCAGCCCCCGATTGGGGGCGACACTCCACCATAGATCACTCCCCTGTGAGGAACTACTGTCTTCACGCA GAAAGCGTCTAGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCCA TAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGATCAACCCG CTCAATGCCTGGAGATTTGGGCGTGCCCCCGCGAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCC TTGTGGTACTGCCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCATCATGAGCACA AATCCTAAACCTCAAAGAAAAACCAAACGTAACACCAACCGCCGCCCACAGGACGTTAAGTTCCCGGGCG GTGGTCAGATCGTTGGTGGAGTTTACCTGTTGCCGCGCAGGGGCCCCAGGTTGGGTGTGCGCGCGACTAG GAAGACTTCCGAGCGGTCGCAACCTCGTGGAAGGCGACAACCTATCCCCAAGGCTCGCCGGCCCGAGGGT AGGACCTGGGCTCAGCCCGGGTACCCTTGGCCCCTCTATGGCAACGAGGGTATGGGGTGGGCAGGATGGC TCCTGTCACCCCGTGGCTCTCGGCCTAGTTGGGGCCCCACAGACCCCCGGCGTAGGTCGCGTAATTTGGG TAAGGTCATCGATACCCTTACATGCGGCTTCGCCGACCTCATGGGGTACATTCCGCTTGTCGGCGCCCCC CTAGGAGGCGCTGCCAGGGCCCTGGCGCATGGCGTCCGGGTTCTGGAGGACGGCGTGAACTATGCAACAG GGAATCTGCCCGGTTGCTCTTTCTCTATCTTCCTCTTAGCTTTGCTGTCTTGTTTGACCATCCCAGCTTC CGCTTACGAGGTGCGCAACGTGTCCGGGATATACCATGTCACGAACGACTGCTCCAACTCAAGTATTGTG TATGAGGCAGCGGACATGATCATGCACACCCCCGGGTGCGTGCCCTGCGTCCGGGAGAGTAATTTCTCCC GTTGCTGGGTAGCGCTCACTCCCACGCTCGCGGCCAGGAACAGCAGCATCCCCACCACGACAATACGACG CCACGTCGATTTGCTCGTTGGGGCGGCTGCTCTCTGTTCCGCTATGTACGTTGGGGATCTCTGCGGATCC GTTTTTCTCGTCTCCCAGCTGTTCACCTTCTCACCTCGCCGGTATGAGACGGTACAAGATTGCAATTGCT CAATCTATCCCGGCCACGTATCAGGTCACCGCATGGCTTGGGATATGATGATGAACTGGTCACCTACAAC GGCCCTAGTGGTATCGCAGCTACTCCGGATCCCACAAGCCGTCGTGGACATGGTGGCGGGGGCCCACTGG GGTGTCCTAGCGGGCCTTGCCTACTATTCCATGGTGGGGAACTGGGCTAAGGTCTTGATTGTGATGCTAC TCTTTGCTGGCGTTGACGGGCACACCCACGTGACAGGGGGAAGGGTAGCCTCCAGCACCCAGAGCCTCGT GTCCTGGCTCTCACAAGGGCCATCTCAGAAAATCCAACTCGTGAACACCAACGGCAGCTGGCACATCAAC AGGACCGCTCTGAATTGCAATGACTCCCTCCAAACTGGGTTCATTGCTGCGCTGTTCTACGCACACAGGT TCAACGCGTCCGGATGTCCAGAGCGCATGGCCAGCTGCCGCCCCATCGACAAGTTCGCTCAGGGGTGGGG TCCCATCACTCACGTTGTGCCTAACATCTCGGACCAGAGGCCTTATTGCTGGCACTATGCACCCCAACCG TGCGGTATTGTACCCGCGTCGCAGGTGTGTGGCCCAGTGTATTGCTTCACCCCGAGTCCTGTTGTGGTGG GGACGACCGACCGTTCCGGAGTCCCCACGTATAGCTGGGGGGAGAATGAGACAGACGTGCTGCTACTCAA CAACACGCGGCCGCCGCAAGGCAACTGGTTCGGCTGTACATGGATGAATAGCACCGGGTTCACCAAGACG TGCGGGGGCCCCCCGTGTAACATCGGGGGGGTTGGCAACAACACCTTGATTTGCCCCACGGATTGCTTCC GAAAGCACCCCGAGGCCACTTACACCAAATGCGGCTCGGGTCCTTGGTTGACACCTAGGTGTCTAGTTGA CTACCCATACAGACTTTGGCACTACCCCTGCACTATCAATTTTACCATCTTCAAGGTCAGGATGTACGTG GGGGGCGTGGAGCACAGGCTCAACGCCGCGTGCAATTGGACCCGAGGAGAGCGCTGTGACCTGGAGGACA GGGATAGATCAGAGCTTAGCCCGCTGCTATTGTCTACAACGGAGTGGCAGGTACTGCCCTGTTCCTTTAC CACCCTACCGGCTCTGTCCACTGGATTGATCCACCTCCATCAGAATATCGTGGACGTGCAATACCTGTAC GGTGTAGGGTCAGTGGTTGTCTCCGTCGTAATCAAATGGGAGTATGTTCTGCTGCTCTTCCTTCTCCTGG CGGACGCGCGCGTCTGTGCCTGCTTGTGGATGATGCTGCTGATAGCCCAGGCTGAGGCCACCTTAGAGAA CCTGGTGGTCCTCAATGCGGCGTCTGTGGCCGGAGCGCATGGCCTTCTCTCCTTCCTCGTGTTCTTCTGC GCCGCCTGGTACATCAAAGGCAGGCTGGTCCCTGGGGCGGCATATGCTCTCTATGGCGTATGGCCGTTGC TCCTGCTCTTGCTGGCTTTACCACCACGAGCTTATGCCATGGACCGAGAGATGGCTGCATCGTGCGGAGG CGCGGTTTTTGTAGGTCTGGTACTCTTGACCTTGTCACCATACTATAAGGTGTTCCTCGCTAGGCTCATA TGGTGGTTACAATATTTTATCACCAGGGCCGAGGCGCACTTGCAAGTGTGGGTCCCCCCTCTTAATGTTC GGGGAGGCCGCGATGCCATCATCCTCCTTACATGCGCGGTCCATCCAGAGCTAATCTTTGACATCACCAA ACTCCTGCTCGCCATACTCGGTCCGCTCATGGTGCTCCAAGCTGGCATAACCAGAGTGCCGTACTTCGTG CGCGCTCAAGGGCTCATTCATGCATGCATGTTAGTGCGGAAGGTCGCTGGGGGTCATTATGTCCAAATGG CCTTCATGAAGCTGGGCGCGCTGACAGGCACGTACATTTACAACCATCTTACCCCGCTACGGGATTGGGC CCACGCGGGCCTACGAGACCTTGCGGTGGCAGTGGAGCCCGTCGTCTTCTCCGACATGGAGACCAAGATC ATCACCTGGGGAGCAGACACCGCGGCGTGTGGGGACATCATCTTGGGTCTGCCCGTCTCCGCCCGAAGGG GAAAGGAGATACTCCTGGGCCCGGCCGATAGTCTTGAAGGGCGGGGGTGGCGACTCCTCGCGCCCATCAC GGCCTACTCCCAACAGACGCGGGGCCTACTTGGTTGCATCATCACTAGCCTTACAGGCCGGGACAAGAAC CAGGTCGAGGGAGAGGTTCAGGTGGTTTCCACCGCAACACAATCCTTCCTGGCGACCTGCGTCAACGGCG TGTGTTGGACCGTTTACCATGGTGCTGGCTCAAAGACCTTAGCCGGCCCAAAGGGGCCAATCACCCAGAT GTACACTAATGTGGACCAGGACCTCGTCGGCTGGCAGGCGCCCCCCGGGGCGCGTTCCTTGACACCATGC ACCTGTGGCAGCTCAGACCTTTACTTGGTCACGAGACATGCTGACGTCATTCCGGTGCGCCGGCGGGGCG ACAGTAGGGGGAGCCTGCTCTCCCCCAGGCCTGTCTCCTACTTGAAGGGCTCTTCGGGTGGTCCACTGCT CTGCCCTTCGGGGCACGCTGTGGGCATCTTCCGGGCTGCCGTATGCACCCGGGGGGTTGCGAAGGCGGTG GACTTTGTGCCCGTAGAGTCCATGGAAACTACTATGCGGTCTCCGGTCTTCACGGACAACTCATCCCCCC CGGCCGTACCGCAGTCATTTCAAGTGGCCCACCTACACGCTCCCACTGGCAGCGGCAAGAGTACTAAAGT GCCGGCTGCATATGCAGCCCAAGGGTACAAGGTGCTCGTCCTCAATCCGTCCGTTGCCGCTACCTTAGGG TTTGGGGCGTATATGTCTAAGGCACACGGTATTGACCCCAACATCAGAACTGGGGTAAGGACCATTACCA CAGGCGCCCCCGTCACATACTCTACCTATGGCAAGTTTCTTGCCGATGGTGGTTGCTCTGGGGGCGCTTA TGACATCATAATATGTGATGAGTGCCATTCAACTGACTCGACTACAATCTTGGGCATCGGCACAGTCCTG GACCAAGCGGAGACGGCTGGAGCGCGGCTTGTCGTGCTCGCCACCGCTACGCCTCCGGGATCGGTCACCG TGCCACACCCAAACATCGAGGAGGTGGCCCTGTCTAATACTGGAGAGATCCCCTTCTATGGCAAAGCCAT CCCCATTGAAGCCATCAGGGGGGGAAGGCATCTCATTTTCTGTCATTCCAAGAAGAAGTGCGACGAGCTC GCCGCAAAGCTGTCAGGCCTCGGAATCAACGCTGTGGCGTATTACCGGGGGCTCGATGTGTCCGTCATAC CAACTATCGGAGACGTCGTTGTCGTGGCAACAGACGCTCTGATGACGGGCTATACGGGCGACTTTGACTC AGTGATCGACTGTAACACATGTGTCACCCAGACAGTCGACTTCAGCTTGGATCCCACCTTCACCATTGAG ACGACGACCGTGCCTCAAGACGCAGTGTCGCGCTCGCAGCGGCGGGGTAGGACTGGCAGGGGTAGGAGAG GCATCTACAGGTTTGTGACTCCGGGAGAACGGCCCTCGGGCATGTTCGATTCCTCGGTCCTGTGTGAGTG CTATGACGCGGGCTGTGCTTGGTACGAGCTCACCCCCGCCCAGACCTCGGTTAGGTTGCGGGCCTACCTG AACACACCAGGGTTGCCCGTTTGCCAGGACCACCTGGAGTTCTGGGAGAGTGTCTTCACAGGCCTCACCC ACATAGATGCACACTTCTTGTCCCAGACCAAGCAGGCAGGAGACAACTTCCCCTACCTGGTAGCATACCA AGCCACGGTGTGCGCCAGGGCTCAGGCCCCACCTCCATCATGGGATCAAATGTGGAAGTGTCTCATACGG CTGAAACCTACGCTGCACGGGCCAACACCCTTGCTGTACAGGCTGGGAGCCGTCCAAAATGAGGTCACCC TCACCCACCCCATAACCAAATACATCATGGCATGCATGTCGGCTGACCTGGAGGTCGTCACTAGCACCTG GGTGCTGGTGGGCGGAGTCCTTGCAGCTCTGGCCGCGTATTGCCTGACAACAGGCAGTGTGGTCATTGTG GGTAGGATTATCTTGTCCGGGAGGCCGGCTATTGTTCCCGACAGGGAGCTTCTCTACCAGGAGTTCGATG AAATGGAAGAGTGCGCCACGCACCTCCCTTACATTGAGCAGGGAATGCAGCTCGCCGAGCAGTTCAAGCA GAAAGCGCTCGGGTTACTGCAAACAGCCACCAAACAAGCGGAGGCTGCTGCTCCCGTGGTGGAGTCCAAG TGGCGAGCCCTTGAGACATTCTGGGCGAAGCACATGTGGAATTTCATCAGCGGGATACAGTACTTAGCAG GCTTATCCACTCTGCCTGGGAACCCCGCAATAGCATCATTGATGGCATTCACAGCCTCTATCACCAGCCC GCTCACCACCCAAAGTACCCTCCTGTTTAACATCTTGGGGGGGTGGGTGGCTGCCCAACTCGCCCCCCCC AGCGCCGCTTCGGCTTTCGTGGGCGCCGGCATCGCCGGTGCGGCTGTTGGCAGCATAGGCCTTGGGAAGG TGCTTGTGGACATTCTGGCGGGTTATGGAGCAGGAGTGGCCGGCGCGCTCGTGGCCTTTAAGGTCATGAG CGGCGAGATGCCCTCTACCGAGGACCTGGTCAATCTACTTCCTGCCATCCTCTCTCCTGGCGCCCTGGTC GTCGGGGTCGTGTGTGCAGCAATACTGCGTCGGCACGTGGGTCCGGGAGAGGGGGCTGTGCAGTGGATGA ACCGGCTGATAGCGTTCGCCTCGCGGGGTAATCACGTTTCCCCCACGCACTATGTGCCTGAGAGCGACGC CGCAGCGCGTGTTACTCAGATCCTCTCCAGCCTTACCATCACTCAGCTGCTGAAAAGGCTCCACCAGTGG ATTAATGAGGACTGCTCCACACCGTGTTCCGGCTCGTGGCTAAGGGATGTTTGGGACTGGATATGCACGG TGTTGACTGACTTCAAGACCTGGCTCCAGTCCAAGCTCCTGCCGCAGCTACCGGGAGTCCCTTTTTTCTC GTGCCAACGCGGGTACAAGGGAGTCTGGCGGGGAGACGGCATCATGCAAACCACCTGCCCATGTGGAGCA CAGATCACCGGACATGTCAAAAACGGTTCCATGAGGATCGTCGGGCCTAAGACCTGCAGCAACACGTGGC ATGGAACATTCCCCATCAACGCATACACCACGGGCCCCTGCACACCCTCTCCAGCGCCAAACTATTCTAG GGCGCTGTGGCGGGTGGCCGCTGAGGAGTACGTGGAGGTCACGCGGGTGGGGGATTTCCACTACGTGACG GGCATGACCACTGACAACGTAAAGTGCCCATGCCAGGTTCCGGCTCCTGAATTCTTCTCGGAGGTGGACG GAGTGCGGTTGCACAGGTACGCTCCGGCGTGCAGGCCTCTCCTACGGGAGGAGGTTACATTCCAGGTCGG GCTCAACCAATACCTGGTTGGGTCACAGCTACCATGCGAGCCCGAACCGGATGTAGCAGTGCTCACTTCC ATGCTCACCGACCCCTCCCACATCACAGCAGAAACGGCTAAGCGTAGGTTGGCCAGGGGGTCTCCCCCCT CCTTGGCCAGCTCTTCAGCTAGCCAGTTGTCTGCGCCTTCCTTGAAGGCGACATGCACTACCCACCATGT CTCTCCGGACGCTGACCTCATCGAGGCCAACCTCCTGTGGCGGCAGGAGATGGGCGGGAACATCACCCGC GTGGAGTCGGAGAACAAGGTGGTAGTCCTGGACTCTTTCGACCCGCTTCGAGCGGAGGAGGATGAGAGGG AAGTATCCGTTCCGGCGGAGATCCTGCGGAAATCCAAGAAGTTCCCCGCAGCGATGCCCATCTGGGCGCG CCCGGATTACAACCCTCCACTGTTAGAGTCCTGGAAGGACCCGGACTACGTCCCTCCGGTGGTGCACGGG TGCCCGTTGCCACCTATCAAGGCCCCTCCAATACCACCTCCACGGAGAAAGAGGACGGTTGTCCTAACAG AGTCCTCCGTGTCTTCTGCCTTAGCGGAGCTCGCTACTAAGACCTTCGGCAGCTCCGAATCATCGGCCGT CGACAGCGGCACGGCGACCGCCCTTCCTGACCAGGCCTCCGACGACGGTGACAAAGGATCCGACGTTGAG TCGTACTCCTCCATGCCCCCCCTTGAGGGGGAACCGGGGGACCCCGATCTCAGTGACGGGTCTTGGTCTA CCGTGAGCGAGGAAGCTAGTGAGGATGTCGTCTGCTGCTCAATGTCCTACACATGGACAGGCGCCTTGAT CACGCCATGCGCTGCGGAGGAAAGCAAGCTGCCCATCAACGCGTTGAGCAACTCTTTGCTGCGCCACCAT AACATGGTTTATGCCACAACATCTCGCAGCGCAGGCCTGCGGCAGAAGAAGGTCACCTTTGACAGACTGC AAGTCCTGGACGACCACTACCGGGACGTGCTCAAGGAGATGAAGGCGAAGGCGTCCACAGTTAAGGCTAA ACTCCTATCCGTAGAGGAAGCCTGCAAGCTGACGCCCCCACATTCGGCCAAATCCAAGTTTGGCTATGGG GCAAAGGACGTCCGGAACCTATCCAGCAAGGCCGTTAACCACATCCACTCCGTGTGGAAGGACTTGCTGG AAGACACTGTGACACCAATTGACACCACCATCATGGCAAAAAATGAGGTTTTCTGTGTCCAACCAGAGAA AGGAGGCCGTAAGCCAGCCCGCCTTATCGTATTCCCAGATCTGGGAGTCCGTGTATGCGAGAAGATGGCC CTCTATGATGTGGTCTCCACCCTTCCTCAGGTCGTGATGGGCTCCTCATACGGATTCCAGTACTCTCCTG GGCAGCGAGTCGAGTTCCTGGTGAATACCTGGAAATCAAAGAAAAACCCCATGGGCTTTTCATATGACAC TCGCTGTTTCGACTCAACGGTCACCGAGAACGACATCCGTGTTGAGGAGTCAATTTACCAATGTTGTGAC TTGGCCCCCGAAGCCAGACAGGCCATAAAATCGCTCACAGAGCGGCTTTATATCGGGGGTCCTCTGACTA ATTCAAAAGGGCAGAACTGCGGTTATCGCCGGTGCCGCGCGAGCGGCGTGCTGACGACTAGCTGCGGTAA CACCCTCACATGTTACTTGAAGGCCTCTGCAGCCTGTCGAGCTGCGAAGCTCCAGGACTGCACGATGCTC GTGAACGGAGACGACCTTGTCGTTATCTGTGAAAGCGCGGGAACCCAAGAGGACGCGGCGAGCCTACGAG TCTTCACGGAGGCTATGACTAGGTACTCTGCCCCCCCCGGGGACCCGCCCCAACCAGAATACGACTTGGA GCTGATAACATCATGTTCCTCCAATGTGTCGGTCGCCCACGATGCATCAGGCAAAAGGGTGTACTACCTC ACCCGTGATCCCACCACCCCCCTCGCACGGGCTGCGTGGGAAACAGCTAGACACACTCCAGTTAACTCCT GGCTAGGCAACATTATCATGTATGCGCCCACTTTGTGGGCAAGGATGATTCTGATGACTCACTTCTTCTC CATCCTTCTAGCACAGGAGCAACTTGAAAAAGCCCTGGACTGCCAGATCTACGGGGCCTGTTACTCCATT GAGCCACTTGACCTACCTCAGATCATTGAACGACTCCATGGCCTTAGCGCATTTTCACTCCATAGTTACT CTCCAGGTGAGATCAATAGGGTGGCTTCATGCCTCAGGAAACTTGGGGTACCACCCTTGCGAGTCTGGAG ACATCGGGCCAGGAGCGTCCGCGCTAGGCTACTGTCCCAGGGGGGGAGGGCCGCCACTTGTGGCAAGTAC CTCTTCAACTGGGCAGTGAAGACCAAACTCAAACTCACTCCAATCCCGGCTGCGTCCCAGCTGGACTTGT CCGGCTGGTTCGTTGCTGGTTACAGCGGGGGAGACATATATCACAGCCTGTGTCGTGCCCGACCCCGCTG GTTCATGCTGTGCCTACTCCTACTTTCTGTAGGGGTAGGCATCTACCTGCTCCCCAACCGATGAACGGGG AGCTAAACACTCCAGGCCAATAGGCCATTTCCTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCT TTTCCTTCTTTTTCCCTTTTTCTTTCTTCCTTCTTTAATGGTGGCTCCATCTTAGCGCTAGTCACGGCTA GCTGTGAAAGGTCCGTGAGCCGCATGACTGCAGAGAGTGCTGATACTGGCCTCTCTGCAGATCATGT (SEQ ID NO: 6688) gi|306286|gb|M96362.1|HPCUNKCDS Hepatitis C virus mRNA, complete cds TGCCAGCCCCCGATTGGGGGCGACACTCCACCATAGATCACTCCCCTGTGAGGAACTACTGTCTTCACGC AGAAAGCGTCTAGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCC ATAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGATCAACCC GCTCAATGCCTGGAGATTTGGGCGTGCCCCCGCGAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGC CTTGTGGTACTGCCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCACCATGAGCAC GAATCCTAAACCTCAAAGAAAAACCAAACGTAACACCAACCGCCGCCCACAGGATATTAAGTTCCCGGGC GGTGGTCAGATCGTTGGTGGAGTTTACTTGTTGCCGCGCAGGGGCCCCAGGTTGGGTGTGCGCGCGACTA GGAAGACTTCCGAGCGGTCGCAACCTCGTGGAAGGCGACAGCCTATCCCCAAGGCTCGCCGGCCCGAGGG CAGGGCCTGGGCTCAGCCCGGGTACCCTTGGCCCCTCTATGGCAATGAGGGCTTGGGGTGGGCAGGATGG CTCCTGTCACCCCGCGGCTCCCGGCCTAGTTGGGGCCCCACGGACCCCCGGCGTAAGTCGCGTAATTTGG GTAAGGTCATCGACACCCTCACATGCGGCTTCGCCGACCTCATGGGGTACATTCCGCTCGTCGGCGCCCC CCTAGGGGGCGTTGCCAGGGCCCTGGCACATGGTGTCCGGGTGCTGGAGGACGGCGTGAACTATGCAACA GGGAATCTGCCCGGTTGCTCTTTCTCTATCTTCCTCTTGGCTCTGCTGTCTTGTTTGACCACCCCAGTTT CCGCTTATGAAGTGCGTAACGCGTCCGGGATGTACCATGTCACGAACGACTGCTCCAACTCAAGCATTGT GTATGAGGCAGCGGACATGATCATGCACACTCCCGGGTGCGTGCCCTGCGTTCGGGAGGACAACTCCTCC CGTTGCTGGGTGGCACTTACTCCCACGCTCGCGGCCAGGAATGCCAGCGTCCCCACTACGACATTGCGAC GCCATGTCGACTTGCTCGTTGGGGTAGCTGCTTTCTGTTCCGCTATGTACGTGGGGGACCTCTGCGGATC TGTTTTCCTTGTTTCCCAGCTGTTCACCTTTTCGCCTCGCCGGCATGAGACGGTACAGGACTGCAACTGC TCAATCTATCCCGGCCGCGTATCAGGTCACCGCATGGCCTGGGATATGATGATGAACTGGTCGCCTACAA CAGCCCTAGTGGTATCGCAGCTACTCCGGATCCCACAAGCTGTCGTGGACATGGTGACAGGGTCCCACTG GGGAATCCTGGCGGGCCTTGCCTACTATTCCATGGTGGGGAACTGGGCTAAGGTCTTAATTGCGATGCTA CTCTTTGCCGGCGTTGACGGAACCACCCACGTGACAGGGGGGGCGCAAGGTCGGGCCGCTAGCTCGCTAA CGTCCCTCTTTAGCCCTGGGCCGGTTCAGCACCTCCAGCTCATAAACACCAACGGCAGCTGGCATATCAA CAGGACCGCCCTGAGCTGCAATGACTCCCTCAACACTGGGTTTGTTGCCGCGCTGTTCTACAAATACAGG TTCAACGCGTCCGGGTGCCCGGAGCGCTTGGCCACGTGCCGCCCCATTGATACATTCGCGCAGGGGTGGG GTCCCATCACTTACACTGAGCCTCATGATTTGGATCAGAGGCCCTATTGCTGGCACTACGCGCCTCAACC GTGTGGTATTGTGCCCACGTTGCAGGTGTGTGGCCCAGTATACTGCTTCACCCCGAGTCCTGTTGCGGTG GGGACTACCGATCGTTTCGGTGCCCCTACATACAGATGGGGGGCAAATGAGACGGACGTGCTGCTCCTTA ACAACGCCGGGCCGCCGCAAGGCAACTGGTTCGGCTGTACATGGATGAATGGCACTGGGTTCACCAAGAC ATGTGGGGGCCCCCCGTGTAACATCGGGGGGGTCGGCAACAATACCTTGACCTGCCCCACGGACTGCTTC CGAAAGCACCCCGGGGCCACTTACACCAAATGCGGTTCGGGGCCTTGGTTAACACCCAGGTGCTTAGTCG ACTACCCGTACAGGCTCTGGCATTACCCCTGCACTGTCAACTTTACCATCTTTAAGGTTAGGATGTACGT GGGGGGCGCGGAGCACAGGCTCGACGCCGCATGCAACTGGACTCGGGGAGAGCGTTGTGACCTGGAGGAC AGGGATAGGTCAGAGCTTAGCCCGCTGCTGCTGTCTACAACAGAGTGGCAGGTACTGCCCTGTTCCTTCA CAACCCTACCGGCTCTGTCCACTGGTTTGATTCATCTCCATCAGAACATCGTGGACATACAATACCTGTA CGGTATAGGGTCGGCGGTTGTCTCCTTTGCGATCAAATGGGAGTATATTGTGCTGCTCTTCCTTCTTCTG GCGGACGCGCGCGTCTGCGCTTGCTTGTGGATGATGCTGCTGGTAGCGCAAGCCGAGGCCGCCTTAGAGA ACCTGGTGGTCCTCAATGCAGCGTCCGTGGCCGGAGCGCATGGCATTCTTTCCTTCATTGTGTTCTTCTG TGCTGCCTGGTACATCAAGGGCAGGCTGGTTCCCGGAGCGGCATACGCCCTCTATGGCGTATGGCCGCTG CTTCTGCTTCTGCTGGCGTTACCACCACGGGCGTACGCCATGGACCGGGAGATGGCCGCATCGTGCGGAG GCGCGGTTTTTGTAGGTCTGGTACTCTTGACCTTGTCACCACACTATAAAGTGTTCCTTGCCAGGTTCAT ATGGTGGCTACAATATCTCATCACCAGAACCGAAGCGCATCTGCAAGTGTGGGTCCCCCCTCTCAACGTT CGGGGGGGTCGCGATGCCATCATCCTCCTCACATGCGTGGTCCACCCAGAGCTAATCTTTGACATCACAA AATATTTGCTCGCCATATTCGGCCCGCTCATGGTGCTCCAGGCCGGCATAACTAGAGTGCCGTACTTCGT GCGCGCACAAGGGCTCATTCGTGCATGCATGTTGGCGCGGAAAGTCGTGGGGGGTCATTACGTCCAAATG GTCTTCATGAAGCTGGCCGCACTAGCAGGTACGTACGTTTATGACCATCTTACTCCACTGCGAGATTGGG CTCACACGGGCTTACGAGACCTTGCAGTGGCAGTAGAGCCCGTTGTCTTCTCTGACATGGAGACCAAAGT CATCACCTGGGGGGCAGACACCGCGGCGTGCGGGGACATCATCTTGGCCCTGCCTGCTTCCGCCCGAAGG GGGAAGGAGATACTTCTGGGACCGGCCGATAGTCTTGAAGGACAGGGGTGGCGACTCCTTGCGCCCATCA CGGCCTACTCCCAACAAACGCGAGGCCTGCTTGGTTGCATCATCACTAGCCTTACAGGCCGGGACAAGAA CCAGGTTGAGGGGGAGGTTCAAGTGGTTTCCACCGCAACACAATCTTTCCTGGCGACCTGCATCAATGGC GTGTGTTGGACTGTCTTCCACGGCGCCGGCTCAAAGACCCTAGCCGGCCCAAAGGGTCCAATCACCCAAA TGTACACCAATGTAGACCAGGACCTTGTTGGCTGGCCGGCACCTCCTGGGGCGCGTTCCCTGACACCATG CACTTGCGGCTCCTCGGACCTTTACCTGGTCACGAGACATGCTGATGTCATTCCGGTGCGCCGGCGGGGT GACGGTAGGGGGAGCCTACTCCCCCCCAGGCCTGTCTCGTACTTGAAGGGCTCCTCGGGTGGTCCACTGC TCTGCCCTTCGGGGCACGCTGTCGGCATACTTCCGGCTGCTGTATGCACCCGGGGGGTTGCCATGGCGGT GGAATTCATACCCGTTGAGTCTATGGAAACTACTATGCGGTCTCCGGTCTTCACGGACAATCCGTCTCCC CCGGCTGTACCGCAGACATTCCAAGTGGCCCACTTACACGCTCCCACCGGCAGCGGCAAGAGCACTAGGG TGCCGGCTGCATATGCAGCCCAAGGGTACAAGGTGCTCGTCCTAAATCCGTCCGTCGCCGCCACCTTGGG TTTTGGGGCGTATATGTCCAAGGCACATGGTATCGACCCCAACCTTAGAACTGGGGTAAGGACCATCACC ACAGGTGCCCCTATCACATACTCCACCTATGGCAAGTTCCTTGCCGACGGTGGCGGCTCCGGGGGCGCCT ATGACATCATAATGTGTGATGAGTGCCACTCAACTGACTCGACTACCATTTATGGCATCGGCACAGTCCT GGACCAAGCGGAGACGGCTGGAGCGCGGCTCGTGGTGCTCTCCACCGCTACGCCTCCGGGATCGGTCACC GTGCCACACCTCAATATCGAGGAGGTGGCCCTGTCTAATACTGGAGAGATCCCCTTCTACGGCAAAGCCA TTCCCATCGAGGCTATCAAGGGGGGAAGGCATCTCATTTTCTGCCATTCCAAGAAGAAGTGTGACGAACT CGCCGCAAAGCTGTCAGGCCTCGGACTCAATGCCGTAGCGTATTACCGGGGTCTTGACGTGTCCGTCATA CCGACCAGCGGAGACGTTGTTGTCGTGGCGACGGACGCTCTAATGACGGGCTTTACCGGCGACTTTGACT CAGTGATCGACTGTAATACGTGTGTCACCCAGACAGTCGATTTCAGCTTGGACCCCACCTTCACCATTGA GACGACGACCGTGCCCCAAGACGCAGTGTCGCGCTCGCAGAGGCGAGGCAGGACTGGTAGGGGCAGGGCT GGCATATACAGGTTTGTGACTCCAGGAGAACGGCCCTCGGGCATGTTCGATTCTTCGGTCCTGTGTGAGT GTTATGACGCGGGTTGTGCGTGGTACGAACTCACGCCCGCTGAGACCTCGGTTAGGTTGCGGGCGTACCT AAACACACCAGGGTTGCCCGTCTGCCAGGACCATCTGGAGTTCTCGGAGGGTGTCTTCACAGGCCTCACC CACATAGATGCCCACTTCTTATCCCAGACTAAACAGGCAGGAGAGAACTTCCCCTACTTGGTAGCATACC AGGCTACAGTGTGCGCCAGGGCTCAAGCCCCACCTCCATCGTGGGATGAAATGTGGAGGTGTCTCATACG GCTGAAACCTACGCTGCACGGGCCAACACCCCTGCTGTATAGGTTAGGAGCCGTCCAAAATGAGGTCACC CTCACACACCCCATAACCAAATTCATCATGACATGTATGTCGGCTGACCTGGAGGTCGTCACCAGCACCT GGGTGCTGGTAGGCGGAGTCCTCGCAGCTCTGGCCGCGTACTGCCTGACAACAGGCAGCGTGGTCATTGT GGGCAGGATCATCCTGTCCGGGAAGCCGGCTATCATCCCCGATAGGGAAGTTCTCTACCAGGAGTTCGAC GAGATGGAGGAGTGTGCCTCACACCTCCCTTACTTCGAACAGGGAATGCAGCTCGCCGAGCAATTCAAAC AGAAGGCGCTCGGGTTGCTGCAAACAGCCACCAAGCAGGCGGAGGCTGCTGCTCCCGTGGTGGAGTCCAA GTGGCGAGCCCTTGAGACCTTCTGGGCGAAGCACATGTGGAACTTCATTAGTGGGATACAGTACTTGGCA GGCTTGTCCACTCTGCCTGGGAACCCCGCAATACGATCACCGATGGCATTCACAGCCTCCATCACCAGCC CGCTCACCACCCAGCATACCCTCTTGTTTAACATCTTGGGGGGATGGGTGGCTGCCCAACTCGCCCCCCC CAGCGCTGCCTCAGCTTTCGTGGGCGCCGGCATCGCTGGAGCCGCTGTTGGCACGATAGGCCTTGGGAAG GTGCTTGTGGACATTCTGGCAGGTTATGGAGCAGGGGTGGCGGGCGCACTTGTGGCCTTTAAGATCATGA GCGGCGAGATGCCTTCAGCCGAGGACATGGTCAACTTACTCCCTGCCATCCTTTCTCCCGGTGCCCTGGT CGTCGGGATTGTGTGTGCAGCAATACTGCGTCGGCATGTGGGCCCAGGGGAAGGGGCTGTGCAGTGGATG AACCGGCTGATAGCGTTCGCCTCGCGGGGTAACCACGTCTCCCCCAGGCACTATGTGCCAGAGAGCGAGC CTGCAGCGCGTGTTACCCAGATCCTTTCCAGCCTCACCATCACTCAGCTGTTGAAGAGACTCCACCAGTG GATTAATGAGGACTGCTCTACGCCATGCTCCAGCTCGTGGCTAAGGGAGATTTGGGACTGGATCTGCACG GTGTTGACTGACTTCAAGACCTGGCTCCAGTCCAAGCTCCTGCCGCGATTACCGGGAGTCCCTTTTTTCT CATGCCAACGCGGGTATAAGGGAGTCTGGCGGGGGGACGGCATCATGCACACCACCTGCCCATGCGGAGC ACAGATCACCGGACACGTCAAAAACGGTTCCATGAGGATCGTTGGGCCTAAAACCTGCAGCAACACGTGG TACGGGACATTCCCCATCAACGCGTACACCACGGGCCCCTGCACACCCTCCCCGGCGCCAAACTATTCCA AGGCATTGTGGAGAGTGGCCGCTGAGGAGTACGTGGAGGTCACGCGGGTGGGAGATTTTCACTACGTGAC GGGCATGACCACTGACAACGTGAAGTGTCCATGCCAGGTTCCGGCCCCCGAATTCTTCACGGAGGTGGAT GGAGTGCGGTTGCACAGGTACGCTCCGGCGTGCAGACCTCTCCTACGGGAGGAGGTCGTATTCCAGGTCG GGCTCCACCAGTACCTGGTCGGGTCACAGCTCCCATGCGAGCCCGAACCGGATGTAGCAGTGCTCACTTC CATGCTCACTGACCCCTCCCACATTACAGCAGAGACGGCTAAGCGTAGGCTGGCCAGGGGGTCTCCCCCC TCCTTGGCCAGCTCTTCAGCTAGCCAGTTGTCTGCGCCTTCCTTGAAGGCGACATGCACTACCCATCATG ACTCCCCGGACGCTGACCTCATTGAGGCCAACCTCTTGTGGCGGCAAGAGATGGGCGGGAACATCACCCG CGTGGAGTCAGAGAATAAGGTGGTAATCCTGGACTCTTTCGACCCGCTCCGAGCGGAGGATGATGAGGGG GAAATATCCGTTCCGGCGGAGATCCTGCGGAAATCCAGGAAATTCCCCCCAGCGCTGCCCATATGGGCGC CGCCGGATTACAACCCTCCGCTGCTAGAGTCCTGGAAGGACCCGGACTACGTTCCTCCGGTGGTACACGG GTGCCCGTTGCCGCCCACCAAGGCCCCTCCAATACCACCTCCACGGAGGAAGAGGACGGTTGTCCTGACA GAATCCACCGTGTCTTCTGCCTTGGCGGAGCTCGCTACTAAGACCTTCGGCAGCTCCGGATCGTCGGCCA TCGACAGCGGTACGGCGACCGCCCCTCCTGACCAAGCCTCCGGTGACGGCGACAGAGAGTCCGACGTTGA GTCGTTCTCCTCCATGCCCCCCCTTGAGGGAGAGCCGGGGGACCCCGATCTCAGCGACGGATCTTGGTCC ACCGTGAGCGAGGAGGCTAGTGAGGACGTCGTCTGCTGTTCGATGTCCTACACATGGACAGGCGCCCTGA TCACGCCATGCGCTGCGGAGGAAAGCAAGTTGCCCATCAACCCGTTGAGCAATTCTTTGCTACGTCACCA CAACATGGTCTATGCTACAACATCCCGCAGCGCAGGCCTGCGGCAGAAGAAGGTCACCTTTGACAGACTG CAAGTCCTGGACGACCACTACCGGGACGTGCTTAAGGAGATGAAGGCGAAGGCGTCCACAGTTAAGGCTA AACTTCTATCTGTAGAAGAAGCCTGCAAACTGACGCCCCCACATTCGGCCAAATCCAAATTTGGCTACGG GGCGAAGGACGTCCGGAGCCTATCCAGCAGGGCCGTTACCCACATCCGCTCCGTGTGGAAGGACCTGCTG GAAGACACTGAAACACCAATTAGCACTACCATCATGGCAAAAAATGAGGTTTTCTGTGTCCAACCAGAGA AGGGAGGCCGCAAGCCAGCTCGCCTTATCGTGTTCCCAGATCTGGGAGTTCGTGTATGCGAGAAGATGGC CCTTTATGACGTGGTCTCCACCCTTCCTCAGGCCGTGATGGGCTCCTCATACGGATTCCAGTACTCTCCT AAGCAGCGGGTCGAGTTCCTGGTGAATACCTGGAAATCAAAGAAATGCCCCATGGGCTTCTCATATGACA CCCGCTGTTTTGACTCAACGGTCACTGAGAATGACATCCGTGTTGAGGAGTCAATTTACCAATGTTGTGA CTTGGCCCCCGAAGCCAAACTGGCCATAAAGTCGCTCACAGAGCGGCTCTATATCGGGGGTCCCCTGACT AATTCAAAAGGGCAGAACTGCGGTTACCGCCGGTGCCGCGCGAGCGGCGTGCTGACGACTAGCTGCGGTA ATACCCTCACATGTTACCTGAAAGCCACTGCGGCCTGTCGAGCTGCGAAGCTCCGGGACTGCACGATGCT CGTGAACGGAGACGACCTTGTCGTTATCTGTGAAAGCGCGGGAACCCAAGAGGATGCGGCGAGCCTACGA GTCTTCACGGAGGCTATGACTAGGTACTCTGCCCCCCCTGGGGACCCGCCTCAACCGGAATACGACTTGG AGTTGATAACATCATGTTCCTCCAATGTGTCGGTCGCACACGATGCATCTGGTAAAAGGGTGTACTACCT CACCCGTGACCCTACCACCCCCCTTGCACGGGCTGCGTGGGAGACAGCTAGACACACTCCAGTCAACTCC TGGCTAGGCAACATCATCATGTATGCGCCCACCTTATGGGCAAGGATGATTCTGATGACTCATTTCTTCT CCATCCTTCTAGCTCAGGAGCAACTTGAAAAAACCCTAGATTGTCAGATCTACGGGGCCTGTTACTCCAT TGAACCACTTGATCTACCTCAGATCATTGAGCGACTCCATGGTCTTAGCGCATTTTCACTCCATAGTTAC TCTCCAGGCGAGATCAATAGGGTGGCTTCATGCCTCAGAAAACTTGGGGTACCACCCTTGCGAGCCTGGA GACATCGGGCCAGAAGTGTCCGCGCTAAGCTACTGTCCCAGGGGGGGAGGGCCGCCACTTGTGGCAAGTA CCTCTTCAACTGGGCGGTGAGGACCAAGCTCAAACTCACTCCAATCCCAGCCGCGTCCCGGTTGGACTTG TCCGGCTGGTTCGTTGCTGGTTACAGCGGGGGAGACATATATCACAGCCTGTCTCGTGCCCGACCCCGCT GGTTCATGTTGTGCCTACTCCTACTTTCCGTGGGGGTAGGCATCTACCTGCTCCCCAACCGATGAATGGG GAGCTAAACACTCCAGGCCAATAGGCCGTTTCTC (SEQ ID NO: 6689) gi|329739|gb|L02836.1|HPCCGENOM Hepatitis C China virus complete genome ATTGGGGGCGACACTCCACCATAGATCACTCCCCTGTGAGGAACTACTGTCTTCACGCAGAAAGCGTCTA GCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCCATAGTGGTCTGC GGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGATCAACCCGCTCAATGCCTG GAGATTTGGGCGTGCCCCCGCGAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCCTTGTGGTACTG CCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCACCATGAGCACGAATCCTAAACC TCAAAGAAAAACCAAACGTAACACCAACCGCCGCCCACAGGACGTCAAGTTCCCGGGCGGTGGTCAGATC GTTGGTGGAGTTTACCTGTTGCCGCGCAGGGGCCCCAGGTTGGGTGTGCGCGCGACTAGGAAGACTTCCG AGCGGTCGCAACCTCGTGGAAGGCGACAACCTATCCCCAAGGCTCGCCGACCCGAGGGCAGGACCTGGGC TCAGCCCGGGTATCCTTGGCCCCTCTATGGCAATGAGGGCTTTGGGTGGGCAGGATGGCTCCTGTCACCC CGCGGCTCCCGGCCTAGTTGGGGCCCCACGGACCCCCGGCGTAGGTCGCGTAATTTGGGTAAGGTCATCG ATACCCTCACATGCGGCTTCGCCGACCTCATGGGGTACATTCCGCTCGTCGGCGCCCCCTTGGGGGGCGC TGCCAGGGCCCTGGCACATGGTGTCCGGGTTCTGGAGGACGGCGTGAACTATGCAACAGGGAATTTGCCC GGTTGCTCTTTCTCTATCTTCCTTTTAGCCTTGCTATCCTGTTTGACCACCCCAGCTTCCGCTTACGAAG TGCGTAACGTGTCCGGGATATACCATGTCACGAACGACTGCTCCAACTCAAGCATTGTGTATGAGGCAGC GGACCTGATCATGCATACCCCTGGGTGCGTGCCCTGCGTTCGGGAAGGCAACTCCTCCCGTTGCTGGGTA GCGCTCACTCCCACGCTCGCGGCCAGGAACGCCACGATCCCCACTGCGACAGTACGACGGCATGTCGATC TGCTCGTTGGGGCGGCTGCTTTCTCTTCCGCCATGTACGTGGGGGATCTCTGCGGATCTGTTTTCCTTGT CTCTCAGCTGTTCACCTTCTCGCCTCGCCGGTATGAGACAATACAGGACTGCAATTGCTCAATCTATCCC GGCCACGTAACAGGTCACCGCATGGCTTGGGATATGATGATGAACTGGTCGCCTACAACAGCTCTAGTGG TGTCGCAGTTACTCCGGATCCCTCAAGCCGTCATGGACATGGTGGTGGGGGCCCACTGGGGAGTCCTGGC GGGCCTTGCCTACTATGCCATGGTGGGGAATTGGGCTAAGGTTTTGATTGTGATGCTACTCTTCGCCGGC GTTGATGGGGATACCTACGCGTCTGGGGGGGCGCAGGGCCGCTCCACCCTCGGGTTCACGTCCCTCTTTA CACCTGGGGCCTCTCAGAAGATCCAGCTTATAAATACCAATGGTAGCTGGCATATCAACAGGACTGCCCT GAACTGCAATGACTCCCTCAATACTGGGTTTCTTGCCGCGCTGTTCTATACACACAGGTTCAACGCGTCC GGATGCGCAGAGCGCATGGCCAGCTGCCGCCCCATTGATACATTCGATCAGGGCTGGGGCCCCATCACTT ATACTGAGCCTGATAGCTCGGACCAGAGGCCTTATTGCTGGCACTACGCGCCTCGAAAGTGCGGCATCGT ACCTGCGTCGGAGGTGTGCGGTCCAGTGTATTGTTTCACCCCAAGCCCTGTCGTCGTGGGGACGACCGAT CGTTTCGGTGTCCCCACATATAGCTGGGGGGAGAATGAGACAGACGTGCTGCTCCTCAACAACACGCGGC CGCCGCAAGGCAACTGGTTTGGCTGTACATGGATGAATGGCACTGGGTTCACCAAGACGTGCGGGGGGCC TCCGTGTAACATCGGGGGGGTCGGCAACAACACTTTGACTTGCCCCACGGATTGCTTTCGGAAGCACCCC GAGGCTACGTATACAAGGTGTGGTTCGGGGCCTTGGCTGACACCTAGGTGCTTAGTTGACTACCCATACA GGCTCTGGCACTACCCCTGCACTGTCAACTTTGCCATCTTCAAAGTTAGGATGTATGTGGGGGGCGTGGA GCACAGGCTCGATGCTGCATGCAACTGGACTCGAGGAGAGCGCTGTAACTTGGAGGACAGGGATAGATCA GAACTCAGCCCGCTGCTACTGTCTACAACAGAGTGGCAGATACTACCCTGCGCCTTCACCACCCTACCGG CTCTGTCCACTGGTTTAATCCATCTCCATCAGAACATCGTGGACGTGCAATACCTGTACGGTATAGGGTC AGCGGTTGCCTCCTTTGCAATTAAATGGGAGTATGTCTTGTTGCTTTTCCTTCTACTAGCAGACGCGCGC GTATGTGCCTGCTTGTGGATGATGCTGCTGATAGCCCAGGCCGAGGCCGCCTTAGAGAACCTGGTGGTCC TCAATGCGGCGTCCGTGGCCGACGCGCATGGCATCCTCTCCTTCCTTGTGTTCTTTTGTGCCGCCTGGTA CATTAAGGGCAGGCTGGTCCCCGGGGCAGCATACGCTTTCTACGGCGTGTGGCCGCTGCTCCTGCTCCTG CTGACATTACCACCACGAGCTTACGCCATGGACCGGGAGATGGCTGCATCGTGCGGAGGCGCGGTTTTTG TAGGTCTGGTATTCCTGACTTTGTCACCATACTACAAGGTGTTCCTCGCTAGGCTCATATGGTGGTTGCA ATACTTCCTCACCATAGCCGAGGCGCACCTGCAAGTGTGGATCCCCCCTCTCAACATTCGAGGGGGCCGC GATGCCATCATCCTCCTCACGTGTGCAATCCACCCAGAGTCAATCTTTGACATCACCAAACTCCTGCTCG CCACGCTCGGTCCGCTCCTGGTGCTTCAGGCTGGCATAACTAGAGTGCCGTACTTTGTGCGCGCTCATGG GCTCATTCGCGCGTGCATGCTATTGCGGAAAGTTGCTGGGGGTCATTATGTCCAAATGGCCTTCATGAAG CTGGGCGCACTGACAGGTACGTACGTCTATAACCATCTTACTCCGCTGCAGTATTGGCCACGCGCGGGTT TACGAGAACTCGCGGTGGCAGTAGAGCCCGTCATCTTCTCTGACATGGAGACCAAGATTATCACCTGGGG GGCAGACACTGCAGCGTGTGGAGACATCATCTTGGGTTTACCCGTCTCCGCCCGAAGGGGAAAGGAGATA CTCCTGGGGCCGGCCGATAGTCTTGAAGGGCAGGGGTGGCGACTCCTTGCGCCCATCACGGCCTACTCCC AACAGACGCGGGGCTTACTTGGTTGCATCATCACTAGCCTCACAGGCCGAGACAAGAACCAGGTCGAGGG GGAGGTTCAAGTGGTCTCCACCGCAACACAATCTTTCCTGGCGACCTGCATCAACGGTGTGTGTTGGACT GTCTATCATGGCGCCGGCTCAAAAACCTTAGCCGGCCCAAAGGGCCCAATCACCCAAATGTACACCAATG TAGACCAGGACCTCGTCGGCTGGCACCGGCCCCCCGGGGCGCGTTCCCTAACACCATGCACCTGCGGCAG CTCGGACCTTTACTTGGTCACGAGACATGCTGATGTCATTCCGGTGCGCCGTCGAGGCGACAGTAGGGGG AGTTTACTCTCCCCCAGGCCTGTCTCCTACCTGAAGGGCTCGTCGGGGGGCCCACTGCTCTGCCCCTTCG GGCACGTTGCAGGCATCTTCCGGGCTGCTGTGTGCACCCGGGGGGTTGCGAAGGCGGTGGATTTTATACC CGTTGAGACCATGGAAACTACCATGCGGTCCCCGGTCTTCACGGACAACTCATCCCCTCCTGCCGTACCG CAGACATTCCAAGTGGCCCATCTACACGCTCCCACTGGCAGCGGCAAAAGCACCAAGGTGCCGGCTGCAT ATGCAGCCCAAGGGTACAAGGTACTTGTCTTGAACCCGTCTGTTGCCGCCACTTTAGGTTTTGGGGCGTA TATGTCTAAGGCACATGGTGTCGACCCCAACATTAGAACCGGGGTAAGGACCATCACCACGGGCGCCCCC ATCACATACTCTACCTATGGCAAGTTCCTTGCTGATGGTGGTTGCTCTGGGGGTGCCTATGACATTATAA TATGTGATGAGTGCCATTCAACTGACTCGACTACCATCTTGGGCATCGGCACGGTCCTGGACCAAGCGGA GACGGCTGGAGCGCGGCTTGTCGTGCTCGCCACCGCTACGCCTCCGGGATCGGTCACCGTGCCACATCCA AACATCGAGGAGGTGGCCCTGTCCAATACTGGAGAGATCCCCTTCTATGGTAAAGCCATCCCCATCGAAG CCATCAGGGGGGGAAGGCATCTCATTTTCTGCCACTCCAAGAAGAAGTGTGACGAGCTTGCTGCAAAGCT ATCATCGCTCGGGCTCAACGCTGTGGCGTACTACCGGGGGCTTGATGTGTCCGTCATACCATCTAGCGGA GACGTCGTTGTCGTGGCAACGGACGCTCTAATGACGGGCTTTACGGGCGACTTTGACTCAGTGATCGACT GTAACACATGTGTTACCCAAACAGTCGATTTCAGCTTGGACCCCACCTTCACCATCGAGACAACGACCGT GCCCCAAGACGCGGTGTCGCGCTCGCAGCGGCGAGGTAGGACTGGCAGGGGTAGGGAAGGCATCTACAGG TTTGTTACTCCAGGAGAACGGCCCTCGGGCATGTTCGACTCCTCAGTCCTGTGTGAGTGCTATGACGCGG GCTGTGCTTGGTACGAGCTCACGCCGGCTGAGACCACGGTTAGGTTGCGGGCTTACCTAAATACACCAGG GTTGCCCGTCTGCCAGGACCATCTGGAGTTCTGGGAGGGCGTCTTCACAGGTCTCACCCATATAGACGCT CACTTTCTGTCCCAGACCAAGCAAGCAGGAGACAACTTCCCCTACCTGGTAGCATACCAAGCTACAGTGT GTGCCAAGGCTCAGGCCCCACCTCCATCGTGGGATCAAATGTGGAAGTGCCTCACACGGGTAAAGCCTAC GCTGCAGGGACCAACACCCCTGCTGTATAGGCTAGGAGCCGTCCAAAATGAGGTCACCCTCACACACCCC ATAACTAAATACATCATGACATGCATGTCGGCTGACCTGGAGGTCGTCACCAGCACCTGGGTGCTGGTGG GCGGAGTCCTTGCAGCTCTGGCCGCGTATTGCCTGACAACGGGCAGCGTGGTCATTGTGGGTAGGATTGT CTTGTCCGGAAGTCCGGCTATTGTTCCTGACAGGGAAGTTCTTTACCAAGACTTCGACGAGATGGAAGAG TGTGCCTCACACCTCCCTTACATCGAACAGGGAATGCAGCTCGCCGAGCAGTTCAAGCAGAAGGCGCTCG GGTTGCTGCAAACAGCCACCAAGCAAGCGGAGGCTGCTGCTCCCGTGGTGGAGTCCAAGTGGCGAGCCCT CGAGACATTTTGGGAAAAACACATGTGGAATTTCATCAGCGGGATACAGTACTTAGCAGGCTTATCCACT CTGCCTGGGAACCCCGCAATGGCATCACTGATGGCATTCACAGCTTCTATCACCAGCCCGCTCACTACCC AACACACCCTCCTGTTTAACATCTTGGGTGGATGGGTGGCTGCCCAACTCGCTCCCCCCAGCGCCGCTTC GGCCTTTGTGGGCGCCGGCATTGCCGGTGCGGCTGTTGGCAGCATAGGCCTTGGGAAGGTGCTTGTGGAC ATCCTGGCGGGTTATGGGGCGGGGGTGGCTGGCGCACTCGTGGCCTTTAAGGTCATGAGTGGCGAAATGC CCTCCACTGAGGACCTGGTTAATTTACTCCCTGCCATCCTCTCTCCTGGTGCCCTAGTCGTCGGGGTCGT GTGCGCAGCAATACTGCGCCGACACGTGGGCCCGGGAGAGGGGGCTGTGCAGTGGATGAACCGGCTGATA GCGTTCGCTTCGCGGGGTAACCATGTCTCCCCCACGCACTATGTGCCTGAAAGTGACGCCGCAGCGCGTG TTACCCAGATCCTCTCCAGCCTTACCATCACTCAGCTGCTGAAAAGACTTCACCAGTGGATTAATGAGGA CTGTTCCACACCATGCTCCGGCTCGTGGCTAAGGGATGTTTGGGATTGGATATGCACGGTGTTGACCGAT TTCAAGACCTGGCTCCAGTCCAAGCTCCTGCCGCGGTTGCCCGGAGTCCCTTTCCTCTCATGCCAACGCG GGTACAAGGGAGTCTGGCGGGGGGACGGTATTATGCAAACCACCTGTCCATGTGGAGCACAGATTACTGG ACATGTCAAAAACGGTTCCATGAGAATCGTTGGGCCTAAGACTTGTAGCAACACGTGGCATGGAACATTC CCCATCAACGCGTACACCACGGGCCCCTGCACACCCTCCCCGGCGCCGAACTATTCCAGGGCGCTGTGGC GGGTGGCTCCTGAGGAGTACGTGGAGGTTACGCGGGTGGGGGATTTCCACTACGTGACGGGCATGACCAC CGACAACGTGAAATGCCCATGCCAAGTCCCGGCCCCTGAATTCTTCACGGAGGTGGATGGAGTACGGCTG CACAGGTACGCTCCGGCGTGCAAACCTCTCCTACGGGAGGAGGTCGTGTTCCAGGTCGGGCTCAACCAAT ACCTGGTTGGATCACAGCTCCCATGCGAGCCCGAGCCGGACGTAACAGTGCTCACTTCCATGCTTACCGA CCCCTCCCACATCACAGCAGAGACGGCCAAGCGTAGGCTGGCCAGGGGGTCTCCCCCCTCCTTGGCCAGC TCTTCAGCTAGCCAATTGTCTGCGCCTTCTTTGAAGGCGACATGTACTACCCATCATGACTCCCCGGACG CCGACCTCATTGAGGCCAACCTCCTGTGGCGGCAGGAGATGGGCGGAAACATCACCCGTGTGGAGTCAGA AAATAAGGTAGTGATCCTGGACTCTTTCGACCCGCTTCGGGCGGAGGAGGACGAGAGGGAAGTATCCGTT GCGGCGGAGATCCTGCGGAAATCCAGGAAGTTCCCCTCAGCGCTGCCCATATGGGCACGCCCAGACTACA ACCCTCCACTGCTAGAGTCCTGGAAGGACCCAGATTATGTCCCTCCGGTGGTACACGGGTGCCCGTTGCC GCCTACCACGGCCCCTCCAGTACCACCTCCACGGAGAAAAAGGACGGTCGTCCTAACAGAGTCATCCGTG TCTTCTGCCTTGGCGGAGCTCGCTACTAAGACCTTCGGCAGCTCTGAATCGTCGGCCGTCGACAGCGGCA CGGCGACTGCCCCTCCTGACGAGGCCTCCGGCGGCGGCGACAAAGGATCCGACGTTGAGTCGTACTCCTC CATGCCCCCCCTTGAGGGAGAGCCGGGGGACCCCGACCTCAGCGACGGGTCCTGGTCTACCGTGAGTGAG GAGGCCAGTGAGGACGTCGTCTGCTGCTCAATGTCCTATACATGGACAGGCGCCTTGATCACGCCATGTG CTGCGGAGGAGAGCAAGCTGCCCATCAACCCGCTGAGCAACTCCTTGCTGCGTCACCACAACATGGTCTA TGCTACAACATCCCGCAGTGCAAGCCTACGGCAGAAGAAGGTCGCTTTTGACAGAATGCAAGTCCTGGAC GACCACTACCGGGACGTGCTCAAGGAGATGAAGGCGAAGGCGTCCACAGTTAAGGCTAAACTCCTATCCA TAGAAGAGGCCTGCAAGCTGACGCCCCCACATTCAGCCAAATCCAAATTTGGCTATGGGGCAAAAGACGT CCGGAACCTATCCAGCAAGGCCGTTAACCACATCCGCTCCGTGTGGAAGGACTTGTTGGAAGACAATGAG ACACCAATCAATACCACCATCATGGCAAAAAATGAGGTTTTCTGCGTCCAACCAGAGAAAGGAGGCCGTA AGCCAGCTCGCCTTATCGTATTCCCAGACTTGGGAGTCCGTGTGTGCGAGAAGATGGCCCTTTATGACGT GGTCTCCACCCTTCCTCAGCCCGTGATGGGCTCCTCATACGGATTCCAGTACTCTCCTGGGCAGCGGGTC GAATTCCTGCTAAATGCCTGGAAATCAAAGGAAAACCCTATGGGCTTCTCATATGACACCCGCTGTTTTG ACTCAACGGTCACTCAGAACGACATCCGTGTTGAGGAGTCAATTTACCAATGTTGTGACTTGGCCCCCGA GGCCAGACGGGCCATAAAGTCGCTCACAGAGCGGCTCTATATCGGGGGTCCCCTGACTAATTCAAAAGGG CAGAACTGCGGTTATCGCCGGTGCCGCGCAAGTGGCGTGCTGACGACCAGCTGCGGTAATACCCTTACAT GTTACTTGAAGGCCTCTGCGGCCTGTCGAGCTGCGAAGCTGCAGGACTGCACGATGCTCGTGAACGGAGA CGACCTTGTCGTTATCTGTGAAAGCGCGGGAACTCAAGAGGATGCGGCGAGCCTACGAGTCTTCACGGAG GCTATGACTAGGTACTCTGCCCCCCCTGGGGACCTGCCCCAACCAGAATACGACTTGGAGCTAATAACAT CATGCTCCTCCAATGTGTCAGTCGCCCACGATGCATCTGGCAAAAGGGTGTACTACCTCACCCGTGACCC CACCATCCCCCTCGCGCGGGCTGCGTGGGAGACAGCTAGACACACTCCAGTCAACTCCTGGCTAGGCAAC ATCATCATGTATGCGCCCACTCTATGGGCAAGGATGATTCTGATGACTCACTTCTTCTCCATCCTTCTAG CTCAGGAGCAACTTGAGAAAGCCCTGGATTGCCAAATCTACGGGGCCTACTACTCCATTGAGCCACTTGA CCTACCTCAGATCATTGAACGACTCCATGGCCTTAGCGCATTTTCACTCCATAGTTACTCTCCAGGTGAG ATCAATAGGGTGGCGTCATGTCTCAGGAAACTTGGGGTACCACCCTTGCGAGTCTGGAGACATCGGGCCA GAAGCGTCCGCGCTAAGCTACTGTCCCAGGGGGGGAGGGCCGCCACTTGTGGCAAGTACCTCTTCAACTG GGCAGTAAAGACCAAGCTTAAACTCACTCCAATCCCGGCTGCGTCCCGGTTGGACTTGTCCGGCTGGTTC GTTGCTGGTTACAGCGGGGGAGACATATATCACAGCCTGTCTCGTGCCCGACCCCGTTGGTTCATGTTGT GCCTACTCCTACTTTCTGTAGGGGTAGGCATCTACCTGCTCCCCAACCGATGAACGGGGAGATAAACACT CCAGGCCAATAGGCCATCCC (SEQ ID NO: 6690) gi|15422182|gb|AY051292.1|Hepatitis C virus from India polyprotein mRNA, complete cds GCCAGCCCCCTGATGGGGGCGACACTCCACCATAGATCACTCCCCTGTGAGGAACTACTGTCTTCACGCA GAAAGCGTCTAGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCCA TAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGATCAACCCG CTCAATGCCTGGAGATTTGGGCGTGCCCCCGCAAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCC TTGTGGTACTGCCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCACCATGAGCACG AATCCTAAACCTCAAAGAAAAACCAAACGTAACACCAACCGACGCCCACAGAACGTTAAGTTCCCGGGTG GCGGCCAGATCGTTGGCGGAGTTTGCTTGTTGCCGCGCAGGGGTCCCAGAGTGGGTGTGCGCGCGACGAG GAAGACTTCCGAGCGGTCACAACCTCGCGGAAGGCGTCAGCCTATTCCCAAGGCCCGCCGACCCGAGGGC AGGTCCTGGGCGCAGCCCGGGTACCCTTGGCCCCTCTATGGCAACGAGGGCTGTGGGTGGGCAGGATGGC TCTTGTCCCCCCGCGGCTCCCGGCCTAGTCGGGGCCCCTCTGACCCCCGGCGCAGGTCACGCAATTTGGG TAAGGTCATCGATACCCTCACGTGTGGCTTCGCCGACCTCATGGGGTACATCCCGCTCGTCGGTGCTCCT CTAGGGGGCGCTGCTAGGGCTCTGGCACATGGTGTTAGGGTTCTAGAAGACGGCGTAAATTACGCAACAG GGAACCTTCCTGGTTGCTCTTTTTCTATCTTCTTGCTTGCTCTTCTCTCCTGCTTGACAGTCCCTGCTTC GGCCGTCGAAGTGCGCAACTCTTCGGGGATCTACCATGTCACCAATGATTGCCCCAATGCGTCTGTTGTG TACGAGACAGATAGCTTGATCATACATCTGCCCGGGTGTGTGCCCTGCGTACGCGAGGGCAACGCTTCGA GGTGCTGGGTCTCCCTTAGTCCTACTGTTGCCGCTAAGGATCCGGGCGTCCCCGTCAACGAGATTCGGCG TCACGTCGACCTGATTGTCGGGGCCGCTGCATTCTGTTCGGCTATGTATGTAGGGGACTTATGCGGTTCC ATCTTCCTCGTTGGCCAGCTTTTCACCCTCTCCCCTAGGCGCCACTGGACAACACAAGACTGTAATTGCT CCATCTACCCAGGACATGTGACAGGCCATCGAATGGCTTGGGACATGATGATGAATTGGTCACCTACTGG CGCTTTGGTGGTAGCGCAGCTACTCCGGATCCCACAAGCCGTCTTGGATATGATAGCCGGTGCCCACTGG GGTGTCCTAGCGGGCCCGGCATACTACTCCATGGTGGGGAACTGGGCTAAGGTTTTGGTTGTGCTACTGC TCTTCGCTGGCGTCGATGCAACCACCCAAGTCACAGGTGGCACCGCGGGCCGTAATGCATATAGATTGGC TAGCCTCTTCTCCACCGGCCCCAGCCAAAATATCCAGCTCATAAACTCCAATGGCAGCTGGCACATTAAC AGGACTGCCCTGAATTGCAATGACAGCCTGCACACCGGCTGGGTAGCAGCGCTGTTCTACTCCCACAAGT TCAACTCTTCGGGGCGTCCTGAGAGGATGGCTAGTTGTCGGCCTCTTACCGCCTTCGACCAAGGGTGGGG GCCCATCACTTACGGGGGGAAAGCTAGTAACGACCAGCGGCCGTATTGCTGGCACTATGCCCCACGCCCG TGCGGTATCGTGCCGGCGAAAGAGGTTTGCGGGCCTGTATACTGTTTCACACCCAGTCCCGTGGTAGTGG GGACGACGGACAAGTACGGCGTTCCTACCTACACATGGGGCGAGAATGAGACGGATGTACTGCTCCTTAA CAACTCTAGGCCGCCAATAGGGAATTGGTTCGGGTGTACGTGGATGAATTCCACTGGTTTCACCAAGACG TGCGGGGCTCCTGCCTGTAACGTCGGCGGGAGCGAGACCAACACCCTGTCGTGCCCCACAGATTGCTTCC GCAGACATCCGGACGCAACATACGCTAAGTGCGGCTCTGGCCCTTGGCTTAACCCTCGATGCATGGTGGA CTACCCTTACAGGCTCTGGCACTATCCCTGCACAGTCAATTACACCATATTCAAGATCAGGATGTTCGTG GGCGGGATTGAGCACAGGCTCACCGCCGCGTGCAACTGGACGCGGGGAGAGCGCTGCGACTTGGACGACA GGGATCGTGCCGAGTTGAGCCCGCTGTTGCTGTCCACCACGCAATGGCAGGTCCTCCCCTGCTCATTCAC AACGCTGCCCGCCCTGTCAACTGGCCTAATACATCTCCACCAGAACATCGTGGACGTGCAGTACCTCTAC GGGTTGAGCTCGGTAGTTACATCCTGGGCCATAAGGTGGGAGTATGTCGTGCTCCTTTTCTTGCTGTTAG CAGATGCCCGCATTTGTGCCTGCCTTTGGATGATGCTTCTCATATCCCAGGTAGAGGCGGCGCTGGAGAA CCTGATAGTCCTCAACGCTGCTTCCCTGGCTGGGACACACGGCATCGTCCCTTTCTTCATCTTTTTTTGT GCAGCCTGGTATCTGAAAGGCAAGTGGGCCCCTGGACTCGTCTACTCCGTCTACGGAATGTGGCCGCTGC TCCTGCTTCTCCTGGCGTTGCCCCAACGGGCGTACGCCTTGGATCAGGAGTTGGCCGCGTCGTGTGGGGC CGTGGTCTTCATCAGCCTAGCGGTACTTACCCTGTCGCCGTACTACAAACAGTACATGGCCCGCGGCATC TGGTGGCTGCAGTACATGCTGACCAGAGCGGAGGCGCTCCTGCACGTCTGGGTCCCCTCGCTCAACGCCC GGGGAGGGCGTGATGGTGCCATACTGCTCATGTGTGTGCTCCACCCGCACTTGCTCTTTGACATCACCAA AATCATGCTGGCCATTCTCGGGCCCCTGTGGATCTTGCAGGCCAGTCTGCTCAGGGTGCCGTACTTCGTG CGCGCCCACGGTCTCATTAGGCTCTGCATGCTGGTGCGCAAAACAGCGGGCGGTCACTATGTGCAGATGG CTCTGTTGAAGCTGGGGGCACTTACTGGCACTTACATTTACAACCACCTTTCCCCACTCCAAGACTGGGC TCATGGCAGCTTGCGTGATCTAGCGGTGGCCACCGAGCCCGTCATCTTCTCCCGGATGGAGATCAAGACT ATCACCTGGGGGGCAGACACCGCGGCCTGTGGAGACATCATCAACGGGCTGCCTGTTTCTGCTCGGAGGG GGAGAGAGGTGTTGTTGGGACCAGCCGATGCCCTGACTGACAAGGGATGGAGGCTTTTAGCCCCCATCAC AGCTTACGCCCAACAGACACGAGGTCTCTTGGGCTGTATTGTCACCAGCCTCACCGGTCGGGACAAAAAT CAAGTGGAGGGGGAAATCCAGATTGTGTCTACCGCAACCCAGACGTTCTTGGCCACTTGCATCAACGGAG CTTGCTGGACTGTTTATCATGGGGCCGGATCGAGGACCATCGCTTCGGCGTCGGGTCCTGTGGTCCGGAT GTACACCAATGTGGACCAGGATTTGGTGGGCTGGCCAGCGCCTCAGGGAGCGCGCTCCCTGACGCCGTGC ACGTGCGGTGCCTCGGATCTGTACTTGGTCACGAGGCACGCGGATGTCATCCCAGTGCGGCGTCGAGGCG ATAACAGGGGAAGCTTGCTTTCTCCCCGGCCCATCTCATACCTAAAAGGATCCTCGGGAGGCCCTCTGCT CTGCCCCATGGGACATGTCGCGGGCATTTTTAGGGCCGCGGTGTGCACCCGTGGGGTTGCAAAGGCGGTC GACTTTGTGCCCGTTGAGTCCTTAGAGACCACCATGAGGTCCCCAGTGTTTACTGACAATTCCAGCCCTC CAACAGTGCCCCAGAGTTACCAGGTGGCACATCTACATGCACCCACTGGGAGTGGCAAGAGCACGAAGGT GCCGGCCGCTTACGCAGCTCAAGGGTACAAGGTACTTGTGCTGAACCCGTCTGTTGCTGCCACCTTAGGG TTCGGTGCTTATATGTCAAAGGCCCATGGGATTGACCCAAACGTCAGGACCGGCGTGAGGACCATTACCA CAGGCTCCCCCATCACCTACTCCACCTACGGGAAATTTTTGGCTGATGGCGGATGCCCAGGAGGTGCGTA CGACATCATAATATGTGACGAATGTCACTCAGTGGACGCCACCTCGATTCTGGGCATAGGGACCGTCTTG GACCAAGCGGAGACGGCGGGGGTTAGGCTCACTGTCCTTGCCACCGCTACACCACCTGGCTTGGTCACCG TGCCACATTCCAACATCGAGGAAGTTGCACTGTCCGCTGACGGGGAGAAACCATTTTATGGTAAGGCCAT CCCCCTAAACTACATCAAGGGGGGGAGGCATCTCATTTTCTGTCATTCCAAGAAGAAGTGCGACGAGCTC GCTGCAAAGCTGGTCGGTCTGGGCGTCAACGCGGTGGCCTTTTACCGTGGCCTCGACGTATCTGTCATTC CAACTACAGGAGACGTCGTTGTTGTAGCGACCGACGCCTTGATGACTGGCTTCACCGGCGATTTCCACTC TGTGATAGACTGCAACACCTGTGTCGTCCAGACAGTCGACTTCAGCCTAGACCCTATATTCTCTATTGAG ACTTCCACCGTGCCCCAGGACGCCGTGTCCCGCTCCCAACGGAGGGGTAGGACCGGTCGAGGGAAGCATG GTATTTACAGATATGTGTCACCCGGGGAGCGGCCGTCTGGCATGTTCGACTCCGTGGTCCTCTGTGAGTG CTATGACGCGGGTTGTGCTTGGTACGAGCTTACACCCGCCGAGACCACAGTCAGGCTACGGGCATACCTT AACACCCCAGGATTGCCCGTGTGCCAGGACCACTTGGAGTTCTGGGAGAGTGTCTTCACCGGCCTCACCC ACATAGATGCCCACTTCCTGTCCCAGACGAAACAGAGTGGGGAGAACTTCCCCTACCTAGTCGCATACCA AGCCACCGTGTGCGCTAGAGCTAGAGCTCCTCCCCCGTCATGGGACCAAATGTGGAAGTGCCTGATACGG CTCAAGCCCACCCTCACTGGGGCTACCCCATTACTATACAGACTGGGTAGTGTACAGAATGAGATCACCT TAACACACCCAATCACCCAATACATCATGGCTTGCATGTCGGCGGACCTGGAGGTCGTCACTAGCACGTG GGTGTTGGTGGGCGGCGTCCTAGCCGCTTTGGCCGCTTACTGCCTGTCCACAGGCAGCGTGGTCATAGTG GGCAGGATAATCCTAGGTGGGAAGCCGGCAGTCATACCTGACAGGGAGGTTCTCTACCGAGAGTTTGATG AGATGGAGGAGTGCGCCGCCCACGTCCCCTACCTCGAGCAGGGGATGCATTTGGCTGGACAGTTCAAGCA GAAAGCTCTCGGGTTGCTCCAGACAGCATCCAAGCAAGCGGAGACGATCACTCCCACTGTCCGCACCAAC TGGCAGAAACTCGAGTCCTTCTGGGCTAAGCACATGTGGAACTTCGTTAGCGGGATACAATACCTGGCGG GCCTGTCAACGCTGCCCGGGAACCCCGCTATAGCGTCGCTGATGTCGTTTACGGCCGCGGTGACGAGTCC ACTAACCACCCAGCAAACCCTCTTCTTTAACATCTTAGGGGGGTGGGTGGCGGCCCAGCTTGCTTCCCCA GCTGCCGCTACTGCTTTTGTCGGTGCTGGTATTACTGGCGCCGTTGTTGGCAGTGTGGGCCTAGGGAAGG TCCTAGTGGACATTATTGCTGGCTACGGGGCTGGTGTGGCGGGGGCCCTCGTGGCTTTCAAAATCATGAG CGGGGAGACCCCCACCACCGAGGATCTAGTCAACCTTCTGCCTGCCATCCTATCGCCAGGAGCTCTCGTT GTCGGCGTGGTGTGCGCAGCAATACTACGCCGGCACGTGGGCCCTGGCGAGGGCGCCGTGCAGTGGATGA ACCGGCTGATAGCGTTTGCTTCTCGGGGTAACCACGTCTCCCCTACACACTACGTGCCGGAGAGCGACGC GTCGGCTCGTGTCACACAAATTCTCACCAGCCTCACTGTTACTCAGCTTCTGAAAAGGCTCCACGTGTGG ATAAGCTCGGATTGCATCGCCCCGTGTGCTAGTTCTTGGCTTAAAGATGTCTGGGACTGGATATGCGAGG TGCTGAGCGACTTCAAGAATTGGCTGAAGGCCAAACTTGTACCACAACTGCCCGGGATCCCATTCGTATC CTGCCAACGCGGGTACCGTGGGGTCTGGCGGGGCGAGGGCATCGTGCACACTCGTTGCCCGTGTGGGGCC AATATAACTGGACATGTCAAGAACGGTTCGATGAGAATCGTCGGGCCTAAGACTTGCAGCAACACCTGGC GTGGGTCGTTCCCCATTAACGCTTACACTACAGGCCCGTGCACGCCCTCCCCGGCGCCGAACTATACGTT CGCGCTATGGAGGGTGTCTGCAGAGGAGTATGTGGAGGTAAGGCGGCTGGGGGACTTCCATTACGTCACG GGGGTGACCACTGATAAACTCAAGTGTCCATGCCAGGTCCCCTCACCCGAGTTCTTCACAGAGGTGGACG GGGTGCGCCTGCATAGGTACGCCCCCCCCTGCAAACCCCTGCTGCGAGAAGAGGTGACGTTTAGCATCGG GCTCAATGAATACTTGGTGGGGTCCCAGTTGCCCTGCGAGCCCGAGCCAGACGTAGCTGTACTGACATCA ATGCTTACAGACCCCTCCCACATCACTGCAGAGACGGCAGCGCGTAGGCTGAAGCGGGGGTCTCCCCCCT CCCTGGCCAGCTCTTCCGCCAGCCAGCTGTCCGCGCCGTCACTGAAGGCAACATGCACCACTCACCACGA CTCTCCAGACGCTGACCTCATAGAAGCCAACCTCCTGTGGAGACAGGAGATGGGGGGGAACATCACTAGG GTGGAGTCGGAGAACAAGATTGTCGTTCTGGATTCTTTCGACCCGCTCGTAGCGGAGGAGGATGATCGGG AGATCTCTATTCCAGCTGAGATTCTGCGGAAGTTCAAGCAGTTTCCTCCCGCTATGCCCATATGGGCACG GCCAGATTATAATCCTCCCCTTGTGGAACCGTGGAAGCGCCCGGACTATGAGCCACCCTTAGTCCACGGG TGCCCCCTACCACCTCCCAAGCCAACTCCGGTGCCGCCACCCCGGAGAAAGAGGACGGTGGTGCTGGACG AGTCTACAGTATCATCTGCTCTGGCTGAGCTTGCCACTAAGACCTTCGGCAGCTCTACAACCTCAGGCGT GACAAGTGGTGAAGCGACTGAATCGTCCCCGGCGCCCTCCTGCGGCGGTGAGCTGGACTCCGAAGCTGAA TCTTACTCCTCCATGCCCCCTCTCGAGGGGGAGCCGGGGGACCCCGATCTCAGCGACGGGTCTTGGTCTA CCGTGAGCAGTGATGGTGGCACGGAAGACGTTGTGTGCTGCTCGATGTCTTACTCGTGGACGGGCGCTTT AATCACGCCCTGTGCCTCAGAGGAAGCCAAGCTCCCTATCAACGCATTGAGCAACTCGCTGCTGCGCCAC CACAACTTGGTGTATTCCACCACCTCTCGCAGCGCTGGCCAGAGACAGAAAAAAGTCACATTTGACAGAG TGCAAGTCCTGGACGACCATTACCGGGACGTGCTCAAGGAGGCTAAGGCCAAGGCATCCACGGTGAAGGC TAGACTGCTATCCGTTGAGGAAGCGTGTAGCCTGACGCCCCCACACTCCGCCAGATCAAAATTTGGCTAT GGGGCGAAGGATGTCCGAAGCCATTCCAGTAAGGCTATACGCCACATCAACTCCGTGTGGCAGGACCTTC TGGAGGACAATACAACACCCATAGACACTACCATCATGGCAAAGAATGAGGTCTTCTGTGTGAAGCCCGA AAAGGGGGGCCGCAAGCCCGCTCGTCTTATCGTGTACCCCGACCTGGGAGTGCGCGTATGCGAGAAGAGG GCTTTGTATGACGTAGTCAAACAGCTCCCCATTGCCGTGATGGGAGCCTCCTACGGGTTCCAGTACTCAC CAGCGCAGCGGGTCGACTTCCTGCTTAAAGCGTGGAAATCTAAGAAAGTCCCCATGGGGTTTTCCTATGA CACCCGTTGCTTTGACTCAACAGTCACTGAGGCTGATATCCGTACGGAGGAAGACCTCTACCAATCTTGT GACCTGGCCCCTGAGGCTCGCATAGCCATAAGGTCCCTCACAGAGAGGCTTTACATCGGGGGCCCACTCA CCAATTCTAAGGGACAAAACTGCGGCTATCGGCGATGCCGCGCAAGCGGCGTGCTGACCACTAGCTGCGG TAACACCATAACCTGCTTCCTCAAAGCCAGTGCAGCCTGTCGAGCTGCGAAGCTCCAGGACTGCACCATG CTCGTGTGCGGCGACGACCTCGTCGTTATCTGTGAGAGCGCCGGTGTCCAGGAGGACGCTGCGAGCCTGA GAGCCTTCACGGAGGCTATGACCAGGTACTCCGCCCCCCCGGGAGACCCGCCTCAACCAGAATACGACTT GGAGCTTATAACATCCTGCTCCTCCAATGTGTCGGTCGCGCGCGACGGCGCTGGCAAAAGGGTCTATTAT CTGACCCGTGACCCTGAGACTCCCCTCGCGCGTGCCGCTTGGGAGACAGCAAGACACACTCCAGTGAACT CCTGGCTAGGCAACATCATCATGTTTGCCCCCACTCTGTGGGTACGGATGGTCCTCATGACCCATTTTTT CTCCATACTCATAGCTCAGGAGCACCTTGGAAAGGCTCTAGATTGTGAAATCTATGGAGCCGTACACTCC GTCCAACCGTTGGACTTACCTGAAATCATCCAAAGACTCCACAGCCTCAGCGCGTTTTCGCTCCACAGTT ACTCTCCAGGTGAAATCAATAGGGTGGCTGCATGCCTCAGGAAGCTTGGGGTTCCGCCCTTGCGAGCTTG GAGACACCGGGCCCGGAGCGTTCGCGCCACACTCCTATCCCAGGGGGGGAAAGCCGCTATATGCGGTAAG TACCTCTTCAACTGGGCGGTGAAAACCAAACTCAAACTCACTCCATTACCGTCCATGTCTCAGTTGGACT TGTCCAACTGGTTCACGGGCGGTTACAGCGGGGGAGACATTTATCACAGCGTGTCTCATGCCCGGCCCCG TTTGTTCCTCTGGTGCCTACTCCTACTTTCAGTAGGGGTAGGCATCTATCTCCTTCCCAACCGATAGACG GNTGGGCAACCACTCCGGGTCTTTAGGCCCTATTTAAACACTCCAGGCCTTTAGGCCCCGT (SEQ ID NO: 6691) gi|23510419|ref|NM_000043.3| Homo sapiens tumor necrosis factor receptor superfamily, member 6 (TNFRSF6), transcript variant 1, mRNA CCTACCCGCGCGCAGGCCAAGTTGCTGAATCAATGGAGCCCTCCCCAACCCGGGCGTTCCCCAGCGAGGC TTCCTTCCCATCCTCCTGACCACCGGGGCTTTTCGTGAGCTCGTCTCTGATCTCGCGCAAGAGTGACACA CAGGTGTTCAAAGACGCTTCTGGGGAGTGAGGGAAGCGGTTTACGAGTGACTTGGCTGGAGCCTCAGGGG CGGGCACTGGCACGGAACACACCCTGAGGCCAGCCCTGGCTGCCCAGGCGGAGCTGCCTCTTCTCCCGCG GGTTGGTGGACCCGCTCAGTACGGAGTTGGGGAAGCTCTTTCACTTCGGAGGATTGCTCAACAACCATGC TGGGCATCTGGACCCTCCTACCTCTGGTTCTTACGTCTGTTGCTAGATTATCGTCCAAAAGTGTTAATGC CCAAGTGACTGACATCAACTCCAAGGGATTGGAATTGAGGAAGACTGTTACTACAGTTGAGACTCAGAAC TTGGAAGGCCTGCATCATGATGGCCAATTCTGCCATAAGCCCTGTCCTCCAGGTGAAAGGAAAGCTAGGG ACTGCACAGTCAATGGGGATGAACCAGACTGCGTGCCCTGCCAAGAAGGGAAGGAGTACACAGACAAAGC CCATTTTTCTTCCAAATGCAGAAGATGTAGATTGTGTGATGAAGGACATGGCTTAGAAGTGGAAATAAAC TGCACCCGGACCCAGAATACCAAGTGCAGATGTAAACCAAACTTTTTTTGTAACTCTACTGTATGTGAAC ACTGTGACCCTTGCACCAAATGTGAACATGGAATCATCAAGGAATGCACACTCACCAGCAACACCAAGTG CAAAGAGGAAGGATCCAGATCTAACTTGGGGTGGCTTTGTCTTCTTCTTTTGCCAATTCCACTAATTGTT TGGGTGAAGAGAAAGGAAGTACAGAAAACATGCAGAAAGCACAGAAAGGAAAACCAAGGTTCTCATGAAT CTCCAACCTTAAATCCTGAAACAGTGGCAATAAATTTATCTGATGTTGACTTGAGTAAATATATCACCAC TATTGCTGGAGTCATGACACTAAGTCAAGTTAAAGGCTTTGTTCGAAAGAATGGTGTCAATGAAGCCAAA ATAGATGAGATCAAGAATGACAATGTCCAAGACACAGCAGAACAGAAAGTTCAACTGCTTCGTAATTGGC ATCAACTTCATGGAAAGAAAGAAGCGTATGACACATTGATTAAAGATCTCAAAAAAGCCAATCTTTGTAC TCTTGCAGAGAAAATTCAGACTATCATCCTCAAGGACATTACTAGTGACTCAGAAAATTCAAACTTCAGA AATGAAATCCAAAGCTTGGTCTAGAGTGAAAAACAACAAATTCAGTTCTGAGTATATGCAATTAGTGTTT GAAAAGATTCTTAATAGCTGGCTGTAAATACTGCTTGGTTTTTTACTGGGTACATTTTATCATTTATTAG CGCTGAAGAGCCAACATATTTGTAGATTTTTAATATCTCATGATTCTGCCTCCAAGGATGTTTAAAATCT AGTTGGGAAAACAAACTTCATCAAGAGTAAATGCAGTGGCATGCTAAGTACCCAAATAGGAGTGTATGCA GAGGATGAAAGATTAAGATTATGCTCTGGCATCTAACATATGATTCTGTAGTATGAATGTAATCAGTGTA TGTTAGTACAAATGTCTATCCACAGGCTAACCCCACTCTATGAATCAATAGAAGAAGCTATGACCTTTTG CTGAAATATCAGTTACTGAACAGGCAGGCCACTTTGCCTCTAAATTACCTCTGATAATTCTAGAGATTTT ACCATATTTCTAAACTTTGTTTATAACTCTGAGAAGATCATATTTATGTAAAGTATATGTATTTGAGTGC AGAATTTAAATAAGGCTCTACCTCAAAGACCTTTGCACAGTTTATTGGTGTCATATTATACAATATTTCA ATTGTGAATTCACATAGAAAACATTAAATTATAATGTTTGACTATTATATATGTGTATGCATTTTACTGG CTCAAAACTACCTACTTCTTTCTCAGGCATCAAAAGCATTTTGAGCAGGAGAGTATTACTAGAGCTTTGC CACCTCTCCATTTTTGCCTTGGTGCTCATCTTAATGGCCTAATGCACCCCCAAACATGGAAATATCACCA AAAAATACTTAATAGTCCACCAAAAGGCAAGACTGCCCTTAGAAATTCTAGCCTGGTTTGGAGATACTAA CTGCTCTCAGAGAAAGTAGCTTTGTGACATGTCATGAACCCATGTTTGCAATCAAAGATGATAAAATAGA TTCTTATTTTTCCCCCACCCCCGAAAATGTTCAATAATGTCCCATGTAAAACCTGCTACAAATGGCAGCT TATACATAGCAATGGTAAAATCATCATCTGGATTTAGGAATTGCTCTTGTCATACCCCCAAGTTTCTAAG ATTTAAGATTCTCCTTACTACTATCCTACGTTTAAATATCTTTGAAAGTTTGTATTAAATGTGAATTTTA AGAAATAATATTTATATTTCTGTAAATGTAAACTGTGAAGATAGTTATAAACTGAAGCAGATACCTGGAA CCACCTAAAGAACTTCCATTTATGGAGGATTTTTTTGCCCCTTGTGTTTGGAATTATAAAATATAGGTAA AAGTACGTAATTAAATAATGTTTTTGGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 6692) gi|35910|emb|X12387.1|HSRCYP3 Human mRNA for cytochrome P-450 (cyp3 locus) GAATTCCCAAAGAGCAACACAGAGCTGAAAGGAAGACTCAGAGGAGAGAGATAAGTAAGGAAAGTAGTGA TGGCTCTCATCCCAGACTTGGCCATGGAAACCTGGCTTCTCCTGGCTGTCAGCCTGGTGCTCCTCTATCT ATATGGAACCCATTCACATGGACTTTTTAAGAAGCTTGGAATTCCAGGGCCCACACCTCTGCCTTTTTTG GGAAATATTTTGTCCTACCATAAGGGCTTTTGTATGTTTGACATGGAATGTCATAAAAAGTATGGAAAAG TGTGGGGCTTTTATGATGGTCAACAGCCTGTGCTGGCTATCACAGATCCTGACATGATCAAAACAGTGCT AGTGAAAGAATGTTATTCTGTCTTCACAAACCGGAGGCCTTTTGGTCCAGTGGGATTTATGAAAAGTGCC ATCTCTATAGCTGAGGATGAAGAATGGAAGAGATTACGATCATTGCTGTCTCCAACCTTCACCAGTGGAA AACTCAAGGAGATGGTCCCTATCATTGCCCAGTATGGAGATGTGTTGGTGAGAAATCTGAGGCGGGAAGC AGAGACAGGCAAGCCTGTCACCTTGAAAGACGTCTTTGGGGCCTACAGCATGGATGTGATCACTAGCACA TCATTTGGAGTGAACATCGACTCTCTCAACAATCCACAAGACCCCTTTGTGGAAAACACCAAGAAGCTTT TAAGATTTGATTTTTTGGATCCATTCTTTCTCTCAATAACAGTCTTTCCATTCCTCATCCCAATTCTTGA AGTATTAAATATCTGTGTGTTTCCAAGAGAAGTTACAAATTTTTTAAGAAAATCTGTAAAAAGGATGAAA GAAAGTCGCCTCGAAGATACACAAAAGCACCGAGTGGATTTCCTTCAGCTGATGATTGACTCTCAGAATT CAAAAGAAACTGAGTCCCACAAAGCTCTGTCCGATCTGGAGCTCGTGGCCCAATCAATTATCTTTATTTT TGCTGGCTATGAAACCACGAGCAGTGTTCTCTCCTTCATTATGTATGAACTGGCCACTCACCCTGATGTC CAGCAGAAACTGCAGGAGGAAATTGATGCAGTTTTACCCAATAAGGCACCACCCACCTATGATACTGTGC TACAGATGGAGTATCTTGACATGGTGGTGAATGAAACGCTCAGATTATTCCCAATTGCTATGAGACTTGA GAGGGTCTGCAAAAAAGATGTTGAGATCAATGGGATGTTCATTCCCAAAGGGTGGGTGGTGATGATTCCA AGCTATGCTCTTCACCGTGACCCAAAGTACTGGACAGAGCCTGAGAAGTTCCTCCCTGAAAGATTCAGCA AGAAGAACAAGGACAACATAGATCCTTACATATACACACCCTTTGGAAGTGGACCCAGAAACTGCATTGG CATGAGGTTTGCTCTCATGAACATGAAACTTGCTCTAATCAGAGTCCTTCAGAACTTCTCCTTCAAACCT TGTAAAGAAACACAGATCCCCCTGAAATTAAGCTTAGGAGGACTTCTTCAACCAGAAAAACCCGTTGTTC TAAAGGTTGAGTCAAGGGATGGCACCGTAAGTGGAGCCTGAATTTTCCTAAGGACTTCTGCTTTGCTCTT CAAGAAATCTGTGCCTGAGAACACCAGAGACCTCAAATTACTTTGTGAATAGAACTCTGAAATGAAGATG GGCTTCATCCAATGGACTGCATAAATAACCGGGGATTCTGTACATGCATTGAGCTCTCTCATTGTCTGTG TAGAGTGTTATACTTGGGAATATAAAGGAGGTGACCAAATCAGTGTGAGGAGGTAGATTTGGCTCCTCTG CTTCTCACGGGACTATTTCCACCACCCCCAGTTAGCACCATTAACTCCTCCTGAGCTCTGATAAGAGAAT CAACATTTCTCAATAATTTCCTCCACAAATTATTAATGAAAATAAGAATTATTTTGATGGCTCTAACAAT GACATTTATATCACATGTTTTCTCTGGAGTATTCTATAGTTTTATGTTAAATCAATAAAGACCACTTTAC AAAAGTATTATCAGATGCTTTCCTGCACATTAAGGAGAATCTATAGAACTGAATGAGAACCAACAAGTAA ATATTTTTGGTCATTGTAATCACTGTTGGCGTGGGGCCTTTGTCAGAACTAGAATTTGATTATTAACATA GGTGAAAGTTAATCCACTGTGACTTTGCCCATTGTTTAGAAAGAATATTCATAGTTTAATTATGCCTTTT TTGATCAGGCACATGGCTCACGCCTGTAATCCTAGCAGTTTGGGAGGCTGAGCCGGGTGGATCGCCTGAG GTCAGGAGTTCAAGACAAGCCTGGCCTACATGGTGAAACCCCATCTCTACTAAAAATACACAAATTAGCT AGGCATGGTGGACTCGCCTGTAATCTCACTACACAGGAGGCTGAGGCAGGAGAATCACTTGAACCTGGGA GGCGGATGTTGAAGTGAGCTGAGATTGCACCACTGCACTCCAGTCTGGGTGAGAGTGAGACTCAGTCTTA AAAAAATATGCCTTTTTGAAGCACGTACATTTTGTAACAAAGAACTGAAGCTCTTATTATATTATTAGTT TTGATTTAATGTTTTCAGCCCATCTCCTTTCATATTTCTGGGAGACAGAAAACATGTTTCCCTACACCTC TTGCTTCCATCCTCAACACCCAACTGTCTCGATGCAATGAACACTTAATAAAAAACAGTCGATTGGTCAA AAAAAAAAAAAAAAAAAAAAAAAGAATTC (SEQ ID NO: 6693) gi|339549|gb|M19154.1|HUMTGFB2A Human transforming growth factor-beta-2 mRNA, complete cds GCCCCTCCCGTCAGTTCGCCAGCTGCCAGCCCCGGGACCTTTTCATCTCTTCCCTTTTGGCCGGAGGAGC CGAGTTCAGATCCGCCACTCCGCACCCGAGACTGACACACTGAACTCCACTTCCTCCTCTTAAATTTATT TCTACTTAATAGCCACTCGTCTCTTTTTTTCCCCATCTCATTGCTCCAAGAATTTTTTTCTTCTTACTCG CCAAAGTCAGGGTTCCCTCTGCCCGTCCCGTATTAATATTTCCACTTTTGGAACTACTGGCCTTTTCTTT TTAAAGGAATTCAAGCAGGATACGTTTTTCTGTTGGGCATTGACTAGATTGTTTGCAAAAGTTTCGCATC AAAAACAACAACAACAAAAAACCAAACAACTCTCCTTGATCTATACTTTGAGAATTGTTGATTTCTTTTT TTTATTCTGACTTTTAAAAACAACTTTTTTTTCCACTTTTTTAAAAAATGCACTACTGTGTGCTGAGCGC TTTTCTGATCCTGCATCTGGTCACGGTCGCGCTCAGCCTGTCTACCTGCAGCACACTCGATATGGACCAG TTCATGCGCAAGAGGATCGAGGCGATCCGCGGGCAGATCCTGAGCAAGCTGAAGCTCACCAGTCCCCCAG AAGACTATCCTGAGCCCGAGGAAGTCCCCCCGGAGGTGATTTCCATCTACAACAGCACCAGGGACTTGCT CCAGGAGAAGGCGAGCCGGAGGGCGGCCGCCTGCGAGCGCGAGAGGAGCGACGAAGAGTACTACGCCAAG GAGGTTTACAAAATAGACATGCCGCCCTTCTTCCCCTCCGAAACTGTCTGCCCAGTTGTTACAACACCCT CTGGCTCAGTGGGCAGCTTGTGCTCCAGACAGTCCCAGGTGCTCTGTGGGTACCTTGATGCCATCCCGCC CACTTTCTACAGACCCTACTTCAGAATTGTTCGATTTGACGTCTCAGCAATGGAGAAGAATGCTTCCAAT TTGGTGAAAGCAGAGTTCAGAGTCTTTCGTTTGCAGAACCCAAAAGCCAGAGTGCCTGAACAACGGATTG AGCTATATCAGATTCTCAAGTCCAAAGATTTAACATCTCCAACCCAGCGCTACATCGACAGCAAAGTTGT GAAAACAAGAGCAGAAGGCGAATGGCTCTCCTTCGATGTAACTGATGCTGTTCATGAATGGCTTCACCAT AAAGACAGGAACCTGGGATTTAAAATAAGCTTACACTGTCCCTGCTGCACTTTTGTACCATCTAATAATT ACATCATCCCAAATAAAAGTGAAGAACTAGAAGCAAGATTTGCAGGTATTGATGGCACCTCCACATATAC CAGTGGTGATCAGAAAACTATAAAGTCCACTAGGAAAAAAAACAGTGGGAAGACCCCACATCTCCTGCTA ATGTTATTGCCCTCCTACAGACTTGAGTCACAACAGACCAACCGGCGGAAGAAGCGTGCTTTGGATGCGG CCTATTGCTTTAGAAATGTGCAGGATAATTGCTGCCTACGTCCACTTTACATTGATTTCAAGAGGGATCT AGGGTGGAAATGGATACACGAACCCAAAGGGTACAATGCCAACTTCTGTGCTGGAGCATGCCCGTATTTA TGGAGTTCAGACACTCAGCACAGCAGGGTCCTGAGCTTATATAATACCATAAATCCAGAAGCATCTGCTT CTCCTTGCTGCGTGTCCCAAGATTTAGAACCTCTAACCATTCTCTACTACATTGGCAAAACACCCAAGAT TGAACAGCTTTCTAATATGATTGTAAAGTCTTGCAAATGCAGCTAAAATTCTTGGAAAAGTGGCAAGACC AAAATGACAATGATGATGATAATGATGATGACGACGACAACGATGATGCTTGTAACAAGAAAACATAAGA GAGCCTTGGTTCATCAGTGTTAAAAAATTTTTGAAAAGGCGGTACTAGTTCAGACACTTTGGAAGTTTGT GTTCTGTTTGTTAAAACTGGCATCTGACACAAAAAAAGTTGAAGGCCTTATTCTACATTTCACCTACTTT GTAAGTGAGAGAGACAAGAAGCAAATTTTTTTTAAAGAAAAAAATAAACACTGGAAGAATTTATTAGTGT TAATTATGTGAACAACGACAACAACAACAACAACAACAAACAGGAAAATCCCATTAAGTGGAGTTGCTGT ACGTACCGTTCCTATCCCGCGCCTCACTTGATTTTTCTGTATTGCTATGCAATAGGCACCCTTCCCATTC TTACTCTTAGAGTTAACAGTGAGTTATTTATTGTGTGTTACTATATAATGAACGTTTCATTGCCCTTGGA AAATAAAACAGGTGTATAAAGTGGAGACCAAATACTTTGCCAGAAACTCATGGATGGCTTAAGGAACTTG AACTCAAACGAGCCAGAAAAAAAGAGGTCATATTAATGGGATGAAAACCCAAGTGAGTTATTATATGACC GAGAAAGTCTGCATTAAGATAAAGACCCTGAAAACACATGTTATGTATCAGCTGCCTAAGGAAGCTTCTT GTAAGGTCCAAAAACTAAAAAGACTGTTAATAAAAGAAACTTTCAGTCAG (SEQ ID NO: 6694) gi|186624|gb|J04111.1|HUMJUNA Human c-jun proto oncogene (JUN), complete cds, clone hCJ-1 CCCGGGGAGGGGACCGGGGAACAGAGGGCCGAGAGGCGTGCGGCAGGGGGGAGGGTAGGAGAAAGAAGGG CCCGACTGTAGGAGGGCAGCGGAGCATTACCTCATCCCGTGAGCCTCCGCGGGCCCAGAGAAGAATCTTC TAGGGTGGAGTCTCCATGGTGACGGGCGGGCCCGCCCCCCTGAGAGCGACGCGAGCCAATGGGAAGGCCT TGGGGTGACATCATGGGCTATTTTTAGGGGTTGACTGGTAGCAGATAAGTGTTGAGCTCGGGCTGGATAA GGGCTCAGAGTTGCACTGAGTGTGGCTGAAGCAGCGAGGCGGGAGTGGAGGTGCGCGGAGTCAGGCAGAC AGACAGACACAGCCAGCCAGCCAGGTCGGCAGTATAGTCCGAACTGCAAATCTTATTTTCTTTTCACCTT CTCTCTAACTGCCCAGAGCTAGCGCCTGTGGCTCCCGGGCTGGTGGTTCGGGAGTGTCCAGAGAGCCTTG TCTCCAGCCGGCCCCGGGAGGAGAGCCCTGCTGCCCAGGCGCTGTTGACAGCGGCGGAAAGCAGCGGTAC CCCACGCGCCCGCCGGGGGACGTCGGCGAGCGGCTGCAGCAGCAAAGAACTTTCCCGGCGGGGAGGACCG GAGACAAGTGGCAGAGTCCCGGAGCGAACTTTTGCAAGCCTTTCCTGCGTCTTAGGCTTCTCCACGGCGG TAAAGACCAGAAGGCGGCGGAGAGCCACGCAAGAGAAGAAGGACGTGCGCTCAGCTTCGCTCGCACCGGT TGTTGAACTTGGGCGAGCGCGAGCCGCGGCTGCCGGGCGCCCCCTCCCCCTAGCAGCGGAGGAGGGGACA AGTCGTCGGAGTCCGGGCGGCCAAGACCCGCCGCCGGCCGGCCACTGCAGGGTCCGCACTGATCCGCTCC GCGGGGAGAGCCGCTGCTCTGGGAAGTGAGTTCGCCTGCGGACTCCGAGGAACCGCTGCGCCCGAAGAGC GCTCAGTGAGTGACCGCGACTTTTCAAAGCCGGGTAGCGCGCGCGAGTCGACAAGTAAGAGTGCGGGAGG CATCTTAATTAACCCTGCGCTCCCTGGAGCGAGCTGGTGAGGAGGGCGCAGCGGGGACGACAGCCAGCGG GTGCGTGCGCTCTTAGAGAAACTTTCCCTGTCAAAGGCTCCGGGGGGCGCGGGTGTCCCCCGCTTGCCAG AGCCCTGTTGCGGCCCCGAAACTTGTGCGCGCACGCCAAACTAACCTCACGTGAAGTGACGGACTGTTCT ATGACTGCAAAGATGGAAACGACCTTCTATGACGATGCCCTCAACGCCTCGTTCCTCCCGTCCGAGAGCG GACCTTATGGCTACAGTAACCCCAAGATCCTGAAACAGAGCATGACCCTGAACCTGGCCGACCCAGTGGG GAGCCTGAAGCCGCACCTCCGCGCCAAGAACTCGGACCTCCTCACCTCGCCCGACGTGGGGCTGCTCAAG CTGGCGTCGCCCGAGCTGGAGCGCCTGATAATCCAGTCCAGCAACGGGCACATCACCACCACGCCGACCC CCACCCAGTTCCTGTGCCCCAAGAACGTGACAGATGAGCAGGAGGGGTTCGCCGAGGGCTTCGTGCGCGC CCTGGCCGAACTGCACAGCCAGAACACGCTGCCCAGCGTCACGTCGGCGGCGCAGCCGGTCAACGGGGCA GGCATGGTGGCTCCCGCGGTAGCCTCGGTGGCAGGGGGCAGCGGCAGCGGCGGCTTCAGCGCCAGCCTGC ACAGCGAGCCGCCGGTCTACGCAAACCTCAGCAACTTCAACCCAGGCGCGCTGAGCAGCGGCGGCGGGGC GCCCTCCTACGGCGCGGCCGGCCTGGCCTTTCCCGCGCAACCCCAGCAGCAGCAGCAGCCGCCGCACCAC CTGCCCCAGCAGATGCCCGTGCAGCACCCGCGGCTGCAGGCCCTGAAGGAGGAGCCTCAGACAGTGCCCG AGATGCCCGGCGAGACACCGCCCCTGTCCCCCATCGACATGGAGTCCCAGGAGCGGATCAAGGCGGAGAG GAAGCGCATGAGGAACCGCATCGCTGCCTCCAAGTGCCGAAAAAGGAAGCTGGAGAGAATCGCCCGGCTG GAGGAAAAAGTGAAAACCTTGAAAGCTCAGAACTCGGAGCTGGCGTCCACGGCCAACATGCTCAGGGAAC AGGTGGCACAGCTTAAACAGAAAGTCATGAACCACGTTAACAGTGGGTGCCAACTCATGCTAACGCAGCA GTTGCAAACATTTTGAAGAGAGACCGTCGGGGGCTGAGGGGCAACGAAGAAAAAAAATAACACAGAGAGA CAGACTTGAGAACTTGACAAGTTGCGACGGAGAGAAAAAAGAAGTGTCCGAGAACTAAAGCCAAGGGTAT CCAAGTTGGACTGGGTTCGGTCTGACGGCGCCCCCAGTGTGCACGAGTGGGAAGGACTTGGTCGCGCCCT CCCTTGGCGTGGAGCCAGGGAGCGGCCGCCTGCGGGCTGCCCCGCTTTGCGGACGGGCTGTCCCCGCGCG AACGGAACGTTGGACTTTCGTTAACATTGACCAAGAACTGCATGGACCTAACATTCGATCTCATTCAGTA TTAAAGGGGGGAGGGGGAGGGGGTTACAAACTGCAATAGAGACTGTAGATTGCTTCTGTAGTACTCCTTA AGAACACAAAGCGGGGGGAGGGTTGGGGAGGGGCGGCAGGAGGGAGGTTTGTGAGAGCGAGGCTGAGCCT ACAGATGAACTCTTTCTGGCCTGCTTTCGTTAACTGTGTATGTACATATATATATTTTTTAATTTGATTA AAGCTGATTACTGTCAATAAACAGCTTCATGCCTTTGTAAGTTATTTCTTGTTTGTTTGTTTGGGTATCC TGCCCAGTGTTGTTTGTAAATAAGAGATTTGGAGCACTCTGAGTTTACCATTTGTAATAAAGTATATAAT TTTTTTATGTTTTGTTTCTGAAAATTCCAGAAAGGATATTTAAGAAAATACAATAAACTATTGGAAAGTA CTCCCCTAACCTCTTTTCTGCATCATCTGTAGATCCTAGTCTATCTAGGTGGAGTTGAAAGAGTTAAGAA TGCTCGATAAAATCACTCTCAGTGCTTCTTACTATTAAGCAGTAAAAACTGTTCTCTATTAGACTTAGAA ATAAATGTACCTGATGTACCTGATGCTATGTCAGGCTTCATACTCCACGCTCCCCCAGCGTATCTATATG GAATTGCTTACCAAAGGCTAGTGCGATGTTTCAGGAGGCTGGAGGAAGGGGGGTTGCAGTGGAGAGGGAC AGCCCACTGAGAAGTCAAACATTTCAAAGTTTGGATTGCATCAAGTGGCATGTGCTGTGACCATTTATAA TGTTAGAAATTTTACAATAGGTGCTTATTCTCAAAGCAGGAATTGGTGGCAGATTTTACAAAAGATGTAT CCTTCCAATTTGGAATCTTCTCTTTGACAATTCCTAGATAAAAAGATGGCCTTTGTCTTATGAATATTTA TAACAGCATTCTGTCACAATAAATGTATTCAAATACCAATAACAGATCTTGAATTGCTTCCCTTTAGTAC TTTTTTGTTCCCAAGTTATATACTGAAGTTTTTATTTTTAGTTGCTGAGGTT (SEQ ID NO: 6695) gi|179982|gb|M57729.1|HUMCCC5 Human complement component C5 mRNA, complete cds CTACCTCCAACCATGGGCCTTTTGGGAATACTTTGTTTTTTAATCTTCCTGGGGAAAACCTGGGGACAGG AGCAAACATATGTCATTTCAGCACCAAAAATATTCCGTGTTGGAGCATCTGAAAATATTGTGATTCAAGT TTATGGATACACTGAAGCATTTGATGCAACAATCTCTATTAAAAGTTATCCTGATAAAAAATTTAGTTAC TCCTCAGGCCATGTTCATTTATCCTCAGAGAATAAATTCCAAAACTCTGCAATCTTAACAATACAACCAA AACAATTGCCTGGAGGACAAAACCCAGTTTCTTATGTGTATTTGGAAGTTGTATCAAAGCATTTTTCAAA ATCAAAAAGAATGCCAATAACCTATGACAATGGATTTCTCTTCATTCATACAGACAAACCTGTTTATACT CCAGACCAGTCAGTAAAAGTTAGAGTTTATTCGTTGAATGACGACTTGAAGCCAGCCAAAAGAGAAACTG TCTTAACCTTCATAGATCCTGAAGGATCAGAAGTTGACATGGTAGAAGAAATTGATCATATTGGAATTAT CTCTTTTCCTGACTTCAAGATTCCGTCTAATCCTAGATATGGTATGTGGACGATCAAGGCTAAATATAAA GAGGACTTTTCAACAACTGGAACCGCATATTTTGAAGTTAAAGAATATGTCTTGCCACATTTTTCTGTCT CAATCGAGCCAGAATATAATTTCATTGGTTACAAGAACTTTAAGAATTTTGAAATTACTATAAAAGCAAG ATATTTTTATAATAAAGTAGTCACTGAGGCTGACGTTTATATCACATTTGGAATAAGAGAAGACTTAAAA GATGATCAAAAAGAAATGATGCAAACAGCAATGCAAAACACAATGTTGATAAATGGAATTGCTCAAGTCA CATTTGATTCTGAAACAGCAGTCAAAGAACTGTCATACTACAGTTTAGAAGATTTAAACAACAAGTACCT TTATATTGCTGTAACAGTCATAGAGTCTACAGGTGGATTTTCTGAAGAGGCAGAAATACCTGGCATCAAA TATGTCCTCTCTCCCTACAAACTGAATTTGGTTGCTACTCCTCTTTTCCTGAAGCCTGGGATTCCATATC CCATCAAGGTGCAGGTTAAAGATTCGCTTGACCAGTTGGTAGGAGGAGTCCCAGTAATACTGAATGCACA AACAATTGATGTAAACCAAGAGACATCTGACTTGGATCCAAGCAAAAGTGTAACACGTGTTGATGATGGA GTAGCTTCCTTTGTGCTTAATCTCCCATCTGGAGTGACGGTGCTGGAGTTTAATGTCAAAACTGATGCTC CAGATCTTCCAGAAGAAAATCAGGCCAGGGAAGGTTACCGAGCAATAGCATACTCATCTCTCAGCCAAAG TTACCTTTATATTGATTGGACTGATAACCATAAGGCTTTGCTAGTGGGAGAACATCTGAATATTATTGTT ACCCCCAAAAGCCCATATATTGACAAAATAACTCACTATAATTACTTGATTTTATCCAAGGGCAAAATTA TCCATTTTGGCACGAGGGAGAAATTTTCAGATGCATCTTATCAAAGTATAAACATTCCAGTAACACAGAA CATGGTTCCTTCATCCCGACTTCTGGTCTATTATATCGTCACAGGAGAACAGACAGCAGAATTAGTGTCT GATTCAGTCTGGTTAAATATTGAAGAAAAATGTGGCAACCAGCTCCAGGTTCATCTGTCTCCTGATGCAG ATGCATATTCTCCAGGCCAAACTGTGTCTCTTAATATGGCAACTGGAATGGATTCCTGGGTGGCATTAGC AGCAGTGGACAGTGCTGTGTATGGAGTCCAAAGAGGAGCCAAAAAGCCCTTGGAAAGAGTATTTCAATTC TTAGAGAAGAGTGATCTGGGCTGTGGGGCAGGTGGTGGCCTCAACAATGCCAATGTGTTCCACCTAGCTG GACTTACCTTCCTCACTAATGCAAATGCAGATGACTCCCAAGAAAATGATGAACCTTGTAAAGAAATTCT CAGGCCAAGAAGAACGCTGCAAAAGAAGATAGAAGAAATAGCTGCTAAATATAAACATTCAGTAGTGAAG AAATGTTGTTACGATGGAGCCTGCGTTAATAATGATGAAACCTGTGAGCAGCGAGCTGCACGGATTAGTT TAGGGCCAAGATGCATCAAAGCTTTCACTGAATGTTGTGTCGTCGCAAGCCAGCTCCGTGCTAATATCTC TCATAAAGACATGCAATTGGGAAGGCTACACATGAAGACCCTGTTACCAGTAAGCAAGCCAGAAATTCGG AGTTATTTTCCAGAAAGCTGGTTGTGGGAAGTTCATCTTGTTCCCAGAAGAAAACAGTTGCAGTTTGCCC TACCTGATTCTCTAACCACCTGGGAAATTCAAGGCATTGGCATTTCAAACACTGGTATATGTGTTGCTGA TACTGTCAAGGCAAAGGTGTTCAAAGATGTCTTCCTGGAAATGAATATACCATATTCTGTTGTACGAGGA GAACAGATCCAATTGAAAGGAACTGTTTACAACTATAGGACTTCTGGGATGCAGTTCTGTGTTAAAATGT CTGCTGTGGAGGGAATCTGCACTTCGGAAAGCCCAGTCATTGATCATCAGGGCACAAAGTCCTCCAAATG TGTGCGCCAGAAAGTAGAGGGCTCCTCCAGTCACTTGGTGACATTCACTGTGCTTCCTCTGGAAATTGGC CTTCACAACATCAATTTTTCACTGGAGACTTGGTTTGGAAAAGAAATCTTAGTAAAAACATTACGAGTGG TGCCAGAAGGTGTCAAAAGGGAAAGCTATTCTGGTGTTACTTTGGATCCTAGGGGTATTTATGGTACCAT TAGCAGACGAAAGGAGTTCCCATACAGGATACCCTTAGATTTGGTCCCCAAAACAGAAATCAAAAGGATT TTGAGTGTAAAAGGACTGCTTGTAGGTGAGATCTTGTCTGCAGTTCTAAGTCAGGAAGGCATCAATATCC TAACCCACCTCCCCAAAGGGAGTGCAGAGGCGGAGCTGATGAGCGTTGTCCCAGTATTCTATGTTTTTCA CTACCTGGAAACAGGAAATCATTGGAACATTTTTCATTCTGACCCATTAATTGAAAAGCAGAAACTGAAG AAAAAATTAAAAGAAGGGATGTTGAGCATTATGTCCTACAGAAATGCTGACTACTCTTACAGTGTGTGGA AGGGTGGAAGTGCTAGCACTTGGTTAACAGCTTTTGCTTTAAGAGTACTTGGACAAGTAAATAAATACGT AGAGCAGAACCAAAATTCAATTTGTAATTCTTTATTGTGGCTAGTTGAGAATTATCAATTAGATAATGGA TCTTTCAAGGAAAATTCACAGTATCAACCAATAAAATTACAGGGTACCTTGCCTGTTGAAGCCCGAGAGA ACAGCTTATATCTTACAGCCTTTACTGTGATTGGAATTAGAAAGGCTTTCGATATATGCCCCCTGGTGAA AATCGACACAGCTCTAATTAAAGCTGACAACTTTCTGCTTGAAAATACACTGCCAGCCCAGAGCACCTTT ACATTGGCCATTTCTGCGTATGCTCTTTCCCTGGGAGATAAAACTCACCCACAGTTTCGTTCAATTGTTT CAGCTTTGAAGAGAGAAGCTTTGGTTAAAGGTAATCCACCCATTTATCGTTTTTGGAAAGACAATCTTCA GCATAAAGACAGCTCTGTACCTAACACTGGTACGGCACGTATGGTAGAAACAACTGCCTATGCTTTACTC ACCAGTCTGAACTTGAAAGATATAAATTATGTTAACCCAGTCATCAAATGGCTATCAGAAGAGCAGAGGT ATGGAGGTGGCTTTTATTCAACCCAGGACACCATCAATGCCATTGAGGGCCTGACGGAATATTCACTCCT GGTTAAACAACTCCGCTTGAGTATGGACATCGATGTTTCTTACAAGCATAAAGGTGCCTTACATAATTAT AAAATGACAGACAAGAATTTCCTTGGGAGGCCAGTAGAGGTGCTTCTCAATGATGACCTCATTGTCAGTA CAGGATTTGGCAGTGGCTTGGCTACAGTACATGTAACAACTGTAGTTCACAAAACCAGTACCTCTGAGGA AGTTTGCAGCTTTTATTTGAAAATCGATACTCAGGATATTGAAGCATCCCACTACAGAGGCTACGGAAAC TCTGATTACAAACGCATAGTAGCATGTGCCAGCTACAAGCCCAGCAGGGAAGAATCATCATCTGGATCCT CTCATGCGGTGATGGACATCTCCTTGCCTACTGGAATCAGTGCAAATGAAGAAGACTTAAAAGCCCTTGT GGAAGGGGTGGATCAACTATTCACTGATTACCAAATCAAAGATGGACATGTTATTCTGCAACTGAATTCG ATTCCCTCCAGTGATTTCCTTTGTGTACGATTCCGGATATTTGAACTCTTTGAAGTTGGGTTTCTCAGTC CTGCCACTTTCACAGTTTACGAATACCACAGACCAGATAAACAGTGTACCATGTTTTATAGCACTTCCAA TATCAAAATTCAGAAAGTCTGTGAAGGAGCCGCGTGCAAGTGTGTAGAAGCTGATTGTGGGCAAATGCAG GAAGAATTGGATCTGACAATCTCTGCAGAGACAAGAAAACAAACAGCATGTAAACCAGAGATTGCATATG CTTATAAAGTTAGCATCACATCCATCACTGTAGAAAATGTTTTTGTCAAGTACAAGGCAACCCTTCTGGA TATCTACAAAACTGGGGAAGCTGTTGCTGAGAAAGACTCTGAGATTACCTTCATTAAAAAGGTAACCTGT ACTAACGCTGAGCTGGTAAAAGGAAGACAGTACTTAATTATGGGTAAAGAAGCCCTCCAGATAAAATACA ATTTCAGTTTCAGGTACATCTACCCTTTAGATTCCTTGACCTGGATTGAATACTGGCCTAGAGACACAAC ATGTTCATCGTGTCAAGCATTTTTAGCTAATTTAGATGAATTTGCCGAAGATATCTTTTTAAATGGATGC TAAAATTCCTGAAGTTCAGCTGCATACAGTTTGCACTTATGGACTCCTGTTGTTGAAGTTCGTTTTTTTG TTTTCTTCTTTTTTTAAACATTCATAGCTGGTCTTATTTGTAAAGCTCACTTTACTTAGAATTAGTGGCA CTTGCTTTTATTAGAGAATGATTTCAAATGCTGTAACTTTCTGAAATAACATGGCCTTGGAGGGCATGAA GACAGATACTCCTCCAAGGTTATTGGACACCGGAAACAATAAATTGGAACACCTCCTCAAACCTACCACT CAGGAATGTTTGCTGGGGCCGAAAGAACAGTCCATTGAAAGGGAGTATTACAAAAACATGGCCTTTGCTT GAAAGAAAATACCAAGGAACAGGAAACTGATCATTAAAGCCTGAGTTTGCTTTC (SEQ ID NO: 6696) gi|189944|gb|L05144.1|HUMPHOCAR Homo sapiens (clone lamda-hPEC-3) phosphoenolpyruvate carboxykinase (PCK1) mRNA, complete cds TGGGAACACAAACTTGCTGGCGGGAAGAGCCCGGAAAGAAACCTGTGGATCTCCCTTCGAGATCATCCAA AGAGAAGAAAGGTGACCTCACATTCGTGCCCCTTAGCAGCACTCTGCAGAAATGCCTCCTCAGCTGCAAA ACGGCCTGAACCTCTCGGCCAAAGTTGTCCAGGGAAGCCTGGACAGCCTGCCCCAGGCAGTGAGGGAGTT TCTCGAGAATAACGCTGAGCTGTGTCAGCCTGATCACATCCACATCTGTGACGGCTCTGAGGAGGAGAAT GGGCGGCTTCTGGGCCAGATGGAGGAAGAGGGCATCCTCAGGCGGCTGAAGAAGTATGACAACTGCTGGT TGGCTCTCACTGACCCCAGGGATGTGGCCAGGATCGAAAGCAAGACGGTTATCGTCACCCAAGAGCAAAG AGACACAGTGCCCATCCCCAAAACAGGCCTCAGCCAGCTCGGTCGCTGGATGTCAGAGGAGGATTTTGAG AAAGCGTTCAATGCCAGGTTCCCAGGGTGCATGAAAGGTCGCACCATGTACGTCATCCCATTCAGCATGG GGCCGCTGGGCTCACCTCTGTCGAAGATCGGCATCGAGCTGACGGATTCGCCCTACGTGGTGGCCAGCAT GCGGATCATGACGCGGATGGGCACGCCCGTCCTGGAAGCACTGGGCGATGGGGAGTTTGTCAAATGCCTC CATTCTGTGGGGTGCCCTCTGCCTTTACAAAAGCCTTTGGTCAACAACTGGCCCTGCAACCCGGAGCTGA CGCTCATCGCCCACCTGCCTGACCGCAGAGAGATCATCTCCTTTGGCAGTGGGTACGGCGGGAACTCGCT GCTCGGGAAGAAGTGCTTTGCTCTCAGGATGGCCAGCCGGCTGGCAGAGGAGGAAGGGTGGCTGGCAGAG CACATGCTGATTCTGGGTATAACCAACCCTGAGGGTGAGAAGAAGTACCTGGCGGCCGCATTTCCCAGCG CCTGCGGGAAGACCAACCTGGCCATGATGAACCCCAGCCTCCCCGGGTGGAAGGTTGAGTGCGTCGGGGA TGACATTGCCTGGATGAAGTTTGACGCACAAGGTCATTTAAGGGCCATCAACCCAGAAAATGGCTTTTTC GGTGTCGCTCCTGGGACTTCAGTGAAGACCAACCCCAATGCCATCAAGACCATCCAGAAGAACACAATCT TTACCAATGTGGCCGAGACCAGCGACGGGGGCGTTTACTGGGAAGGCATTGATGAGCCGCTAGCTTCAGG CGTCACCATCACGTCCTGGAAGAATAAGGAGTGGAGCTCAGAGGATGGGGAACCTTGTGCCCACCCCAAC TCGAGGTTCTGCACCCCTGCCAGCCAGTGCCCCATCATTGATGCTGCCTGGGAGTCTCCGGAAGGTGTTC CCATTGAAGGCATTATCTTTGGAGGCCGTAGACCTGCTGGTGTCCCTCTAGTCTATGAAGCTCTCAGCTG GCAACATGGAGTCTTTGTGGGGGCGGCCATGAGATCAGAGGCCACAGCGGCTGCAGAACATAAAGGCAAA ATCATCATGCATGACCCCTTTGCCATGCGGCCCTTCTTTGGCTACAACTTCGGCAAATACCTGGCCCACT GGCTTAGCATGGCCCAGCACCCAGCAGCCAAACTGCCCAAGATCTTCCATGTCAACTGGTTCCGGAAGGA CAAGGAAGGCAAATTCCTCTGGCCAGGCTTTGGAGAGAACTCCAGGGTGCTGGAGTGGATGTTCAACCGG ATCGATGGAAAAGCCAGCACCAACGTCACGCCCATAGGCTACATCCCCAAGGAGGATGCCCTGAACCTGA AAGGCCTGGGGCACATCAACATGATGGAGCTTTTCAGCATCTCCAAGGAATTCTGGGACAAGGAGGTGGA AGACATCGAGAAGTATCTGGTGGATCAAGTCAATGCCGACCTCCCCTGTGAAATCGAGAGAGAGATCCTT GCCTTGAAGCAAAGAATAAGCCAGATGTAATCAGGGCCTGAGAATAAGCCAGATGTAATCAGGGCCTGAG TGCTTTACCTTTAAAATCATTAAATTAAAATCCATAAGGTGCAGTAGGAGCAAGAGAGGGCAAGTGTTCC CAAATTGACGCCACCTAATAATCATCACCACACCGGGAGCAGATCTGAAGGCACACTTTGATTTTTTTAA GGATAAGAACCACAGAACACTGGGTAGTAGCTAATGAAATTGAGAAGGGAAATCTTAGCATGCCTCCAAA AATTCACATCCAATGCATACTTTGTTCAAATTTAAGGTTACTCAGGCATTGATCTTTTCAGTGTTTTTTC ACTTAGCTATGTGGATTAGCTAGAATGCACACCAAAAAGATACTTGAGCTGTATATATATATGTGTGTGT GTGTGTGTGTGTGTGTGTGTGTGCATGTATGTGCACATGTGTCTGTGTGATATTTGGTATGTGTATTTGT ATGTACTGTTATTCAAAATATATTTAATACCTTTGGAAAATCTTGGGCAAGATGACCTACTAGTTTTCCT TGAAAAAAAGTTGCTTTGTTATTAATATTGTGCTTAAATTATTTTTATACACCATTGTTCCTTACCTTTA CATAATTGCAATATTTCCCCCTTACTACTTCTTGGAAAAAAATTAGAAAATGAAGTTTATAGAAAAG (SEQ ID NO: 6697) gi|6679892|ref|NM_008061.1| Mus musculus glucose-6-phosphatase, catalytic (G6pc), mRNA AGCAGAGGGATCGGGGCCAACCGGGCTTGGACTCACTGCACGGGCTCTGCTGGCAGCTTCCTGAGGTACC AAGGGAGGAAGGATGGAGGAAGGAATGAACATTCTCCATGACTTTGGGATCCAGTCGACTCGCTATCTCC AAGTGAATTACCAAGACTCCCAGGACTGGTTCATCCTTGTGTCTGTGATTGCTGACCTGAGGAACGCCTT CTATGTCCTCTTTCCCATCTGGTTCCATCTTAAAGAGACTGTGGGCATCAATCTCCTCTGGGTGGCAGTG GTCGGAGACTGGTTCAACCTCGTCTTCAAGTGGATTCTGTTTGGACAACGCCCGTATTGGTGGGTCCTGG ACACCGACTACTACAGCAACAGCTCCGTGCCTATAATAAAGCAGTTCCCTGTCACCTGTGAGACCGGACC AGGAAGTCCCTCTGGCCATGCCATGGGCGCAGCAGGTGTATACTATGTTATGGTCACTTCTACTCTTGCT ATCTTTCGAGGAAAGAAAAAGCCAACGTATGGATTCCGGTGTTTGAACGTCATCTTGTGGTTGGGATTCT GGGCTGTGCAGCTGAACGTCTGTCTGTCCCGGATCTACCTTGCTGCTCACTTTCCCCACCAGGTCGTGGC TGGAGTCTTGTCAGGCATTGCTGTGGCTGAAACTTTCAGCCACATCCGGGGCATCTACAATGCCAGCCTC CGGAAGTATTGTCTCATCACCATCTTCTTGTTTGGTTTCGCGCTTGGATTCTACCTGCTACTAAAAGGGC TAGGGGTGGACCTCCTGTGGACTTTGGAGAAAGCCAAGAGATGGTGTGAGCGGCCAGAATGGGTCCACCT TGACACTACACCCTTTGCCAGCCTCTTCAAAAACCTGGGAACCCTCTTGGGGTTGGGGCTGGCCCTCAAC TCCAGCATGTACCGGAAGAGCTGCAAGGGAGAACTCAGCAAGTCGTTCCCATTCCGCTTCGCCTGCATTG TGGCTTCCTTGGTCCTCCTGCATCTCTTTGACTCTCTGAAGCCCCCATCCCAGGTTGAGTTGATCTTCTA CATCTTGTCTTTCTGCAAGAGCGCAACAGTTCCCTTTGCATCTGTCAGTCTTATCCCATACTGCCTAGCC CGGATCCTGGGACAGACACACAAGAAGTCTTTGTAAGGCATGCAGAGTCTTTGGTATTTAAAGTCAACCG CCATGCAAAGGACTAGGAACAACTAAAGCCTCTGAAACCCATTGTGAGGCCAGAGGTGTTGACATCGGCC CTGGTAGCCCTGTCTTTCTTTGCTATCTTAACCAAAAGGTGAATTTTTACAAAGCTTACAGGGCTGTTTG AGGAAAGTGTGAATGCTGGAAACTGAGTCATTCTGGATGGTTCCCTGAAGATTCGCTTACCAGCCTCCTG TCAGATACAGAAGAGCAAGCCCAGGCTAGAGATCCCAACTGAGAATGCTCTTGCGGTGCAGAATCTTCCG GCTGGGAAAAGGAAAAGAGCACCATGCATTTGCCAGGAAGAGAAAGAAGGATCGGGAGGAGGGAGAGTGT TTTATGTATCGAGCAAACCAGATGCAATCTATGTCTAACCGGCTTCAGTTGTGTCTGCGTCTTTAGATAC GACACACTCAATAATAATAATAGACCAACTAGTGTAATGAGTAGCCAGTTAAAGGCGATTAATTCTGCTT CCAGATAGTCTCCACTGTACATAAAAGTCACACTGTGTGCTTGCATTCCTGTATGGTAGTGGTGACTGTC TCTCACACCACCTTCTCTATCACGTCACAGTTTTCTCCTCCTCAGCCTATGTCTGCATTCCCCAGAATTC TCCACTTGTTCCCTGGCCCTGCTGCTGGACCCTGCTGTGTCTGGTAGGCAACTGTTTGTTGGTGCTTTTG TAGGGTTAAGTTAAACTCTGAGATCTTGGGCAAAATGGCAAGGAGACCCAGGATTCTTCTCTCCAAAGGT CACTCCGATGTTATTTTTGATTCCTGGGGCAGAAATATGACTCCTTTCCCTAGCCCAAGCCAGCCAAGAG CTCTCATTCTTAGAAGAAAAGGCAGCCCCTTGGTGCCTGTCCTCCTGCCTCGGCTGATTTGCAGAGTACT TCTTCAAAAAGAAAAAAATGGTAAAGCTATTTATTAAAAATTCTTTGTTTTTTGCTACAAATGATGCATA TATTTTCACCCACACCAAGCACTTTGTTTCTAATATCTTTGATAAGAAAACTACATGTGCAGTATTTTAT TAAAGCAACATTTTATTTA (SEQ ID NO: 6698) gi|7110682|ref|NM_011044.1| Mus musculus phosphoenolpyruvate carboxykinase 1, cytosolic (Pck1), mRNA ACAGTTGGCCTTCCCTCTGGGAACACACCCTCGGTCAACAGGGGAAATCCGGCAAGGCGCTCAGCGATCT CTGATCCAGACCTTCCAAAAGGAAGAAAGGTGGCACCAGAGTTCCTGCCTCTCTCCACACCATTGCAATT ATGCCTCCTCAGCTGCATAACGGTCTGGACTTCTCTGCCAAGGTTATCCAGGGCAGCCTCGACAGCCTGC CCCAGGCAGTGAGGAAGTTCGTGGAAGGCAATGCTCAGCTGTGCCAGCCGGAGTATATCCACATCTGCGA TGGCTCCGAGGAGGAGTACGGGCAGTTGCTGGCCCACATGCAGGAGGAGGGTGTCATCCGCAAGCTGAAG AAATATGACAACTGTTGGCTGGCTCTCACTGACCCTCGAGATGTGGCCAGGATCGAAAGCAAGACAGTCA TCATCACCCAAGAGCAGAGAGACACAGTGCCCATCCCCAAAACTGGCCTCAGCCAGCTGGGCCGCTGGAT GTCGGAAGAGGACTTTGAGAAAGCATTCAACGCCAGGTTCCCAGGGTGCATGAAAGGCCGCACCATGTAT GTCATCCCATTCAGCATGGGGCCACTGGGCTCGCCGCTGGCCAAGATTGGTATTGAACTGACAGACTCGC CCTATGTGGTGGCCAGCATGCGGATCATGACTCGGATGGGCATATCTGTGCTGGAGGCCCTGGGAGATGG GGAGTTCATCAAGTGCCTGCACTCTGTGGGGTGCCCTCTCCCCTTAAAAAAGCCTTTGGTCAACAACTGG GCCTGCAACCCTGAGCTGACCCTGATCGCCCACCTCCCGGACCGCAGAGAGATCATCTCCTTTGGAAGCG GATATGGTGGGAACTCACTACTCGGGAAGAAATGCTTTGCGTTGCGGATCGCCAGCCGTCTGGCTAAGGA GGAAGGGTGGCTGGCGGAGCATATGCTGATCCTGGGCATAACTAACCCCGAAGGCAAGAAGAAATACCTG GCCGCAGCCTTCCCTAGTGCCTGTGGGAAGACTAACTTGGCCATGATGAACCCCAGCCTGCCCGGGTGGA AGGTCGAATGTGTGGGCGATGACATTGCCTGGATGAAGTTTGATGCCCAAGGCAACTTAAGGGCTATCAA CCCAGAAAACGGGTTTTTTGGAGTTGCTCCTGGCACCTCAGTGAAGACAAATCCAAATGCCATTAAAACC ATCCAGAAAAACACCATCTTCACCAACGTGGCCGAGACTAGCGATGGGGGTGTTTACTGGGAAGGCATCG ATGAGCCGCTGGCCCCGGGAGTCACCATCACCTCCTGGAAGAACAAGGAGTGGAGACCGCAGGACGCGGA ACCATGTGCCCATCCCAACTCGAGATTCTGCACCCCTGCCAGCCAGTGCCCCATTATTGACCCTGCCTGG GAATCTCCAGAAGGAGTACCCATTGAGGGTATCATCTTTGGTGGCCGTAGACCTGAAGGTGTCCCCCTTG TCTATGAAGCCCTCAGCTGGCAGCATGGGGTGTTTGTAGGAGCAGCCATGAGATCTGAGGCCACAGCTGC TGCAGAACACAAGGGCAAGATCATCATGCACGACCCCTTTGCCATGCGACCCTTCTTCGGCTACAACTTC GGCAAATACCTGGCCCACTGGCTGAGCATGGCCCACCGCCCAGCAGCCAAGTTGCCCAAGATCTTCCATG TCAACTGGTTCCGGAAGGACAAAGATGGCAAGTTCCTCTGGCCAGGCTTTGGCGAGAACTCCCGGGTGCT GGAGTGGATGTTCGGGCGGATTGAAGGGGAAGACAGCGCCAAGCTCACGCCCATCGGCTACATCCCTAAG GAAAACGCCTTGAACCTGAAAGGCCTGGGGGGCGTCAACGTGGAGGAGCTGTTTGGGATCTCTAAGGAGT TCTGGGAGAAGGAGGTGGAGGAGATCGACAGGTATCTGGAGGACCAGGTCAACACCGACCTCCCTTACGA AATTGAGAGGGAGCTCCGAGCCCTGAAACAGAGAATCAGCCAGATGTAAATCCCAATGGGGGCGTCTCGA GAGTCACCCCTTCCCACTCACAGCATCGCTGAGATCTAGGAGAAAGCCAGCCTGCTCCAGCTTTGAGATA GCGGCACAATCGTGAGTAGATCAGAAAAGCACCTTTTAATAGTCAGTTGAGTAGCACAGAGAACAGGCTA GGGGCAAATAAGATTGGGAGGGGAAATCACCGCATAGTCTCTGAAGTTTGCATTTGACACCAATGGGGGT TTTGGTTCCACTTCAAGGTCACTCAGGAATCCAGTTCTTCACGTTAGCTGTAGCAGTTAGCTAAAATGCA CAGAAAACATACTTGAGCTGTATATATGTGTGTGAACGTGTCTCTGTGTGAGCATGTGTGTGTGTGTGTG TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTACATGCCTGTCTGTCCCATTGTCCACAGTATATTTAA AACCTTTGGGGAAAAATCTTGGGCAAATTTGTAGCTGTAACTAGAGAGTCATGTTGCTTTGTTGCTAGTA TGTATGTTTAAATTATTTTTATACACCGCCCTTACCTTTCTTTACATAATTGAAATTGGTATCCGGACCA CTTCTTGGGAAAAAAATTACAAAATAAA (SEQ ID NO: 6699)

Example 6 siRNAs Decrease mRNA Levels In Vivo

Male CMV-Luc mice (8-10 weeks old) from Xenogen (Cranbury, N.J.) were administered cholesterol conjugated siRNA (see Table 16).

TABLE 16 Solutions adminstered to mice Group n Injection Mix 1 7 Buffer (PBS [pH 7.4]) 2 8 Cholesterol conjugated siRNA (ALN-3001)

TABLE 17 Test iRNA agents targeting Luciferase siRNA Sequence ALN-1070 5′-GAA CUG UGU GUG AGA GGU CCU-3′ (SEQ ID NO: 6700) 3′-CG CUU GAC ACA CAC UCU CCA GGA-5′ (SEQ ID NO: 6701) ALN-1000 5′-GAA CUG UGU GUG AGA GGU CCU-GS-3′ (SEQ ID NO: 6702) 3′-CG CUU GAC ACA CAC UCU CCA GGA-5′ (SEQ ID NO: 6703) ALN-3000 5′-GAA CUG UGU GUG AGA GGU CCU-3′ (SEQ ID NO: 6704) 3′-Cs¹Gs¹ CUU GAC ACA CAC UCU CCA GGA-5′ (SEQ ID NO: 6705) ALN-3001 5′-GAA CUG UGU GUG AGA GGU CCU-chol.²-3′ (SEQ ID NO: 6706) 3′-Cs¹Gs¹ CUU GAC ACA CAC UCU CCA GGA-5′ (SEQ ID NO: 6707) ¹2′ O—Me group is attached to the nucleotide and the nucleotides have phosphorothioate linkages (indicated by “s”) ²cholesterol is conjugated to the antisense strand via the linker: U-pyrroline carrier-C(O)—(CH₂)₅—NHC(O)-cholesterol (via cholesterol C-3 hydroxyl).

Animals were injected (tail vein) with a volume of 200-250 μl test solution containing buffer or an siRNA solution. Group 1 received buffer and group 2 received cholesterol conjugated siRNA (ALN-3001) at a dose of 50 mg/kg body weight. Twenty-two hours after injection, animals were sacrificed and livers collected. Organs were snap frozen on dry ice, then pulverized in a mortar and pestle.

For Luciferase mRNA analysis (by the QuantiGene Assay (Genospectra, Inc.; Fremont, Calif.)), approximately 10 mg of tissue powder was resuspended in tissue lysis buffer, and processed according to the manufacturer's protocol. Samples of the lysate were hybridized with probes specific for Luciferase or GAPDH (designed using ProbeDesigner software (Genospectra, Inc., Fremont, Calif.) in triplicate, and processed for luminometric analysis. Values for Luciferase were normalized to GAPDH. Mean values were plotted with error bars corresponding to the standard deviation of the Luciferase measurements.

Results indicated that the level of luciferase RNA in animals injected with cholesterol conjugated siRNA was reduced by about 70% as compared to animals injected with buffer (see FIGS. 6A and 6 b).

In Vitro Activity

HeLa cells expressing luciferase were transfected with each of the siRNAs listed in Table 17. ALN-1000 siRNAs were most effective at decreasing luciferase mRNA levels (˜0.6 nM siRNA decreased mRNA levels to about ˜65% the original expression level, and 1.0 nM siRNA decreased levels to about ˜20% the original expression level); ALN-3001 siRNAs were least effective (˜0.6 nM siRNA had a negligible mRNA levels, and 1.0 nM siRNA decreased levels to about ˜40% the original expression level).

Pharmacokinetics/Biodistribution

Pharmacokinetic analyses were performed in mice and rats. Test siRNA molecules were radioactively labeled with ³³P on the antisense strand by splint ligation. Labeled siRNAs (50 mg/kg) were administered by tail vein injection, and plasma levels of siRNA were measured periodically over 24 hrs by scintillation counting. Cholesterol conjugated siRNA (ALN-3001) was discovered to circulate in mouse plasma for a longer period time than unconjugated siRNA (ALN-3000) (FIG. 7). RNAse protection assays indicated that cholesterol-conjugated siRNA (ALN-3001) was detectable in mouse plasma 12 hours after injection, whereas unconjugated siRNA (ALN-3000) was not detectable in mouse plasma within two hours following injection. Similar results were observed in rats.

Mouse liver was harvested at varying time points (ranging from 0.08-24 hours) following injection with siRNA, and siRNA localized to the liver was quantified. Over the time period tested, the amount of cholesterol-conjugated siRNA (ALN-3001) detected in the liver ranged from 14.3-3.55 percent of the total dose administered to the mouse. The amount of unconjugated siRNA (ALN-3000) detected in the liver was lower, ranging from 3.91-1.75 percent of the total dose administered.

Detection of siRNA in Different Tissues

Various tissues and organs (fat, heart, kidney, liver, and spleen) were harvested from two CMV-Luc mice 22 hours following injection with 50 mg/kg ALN-3001. The antisense strand of the siRNA was detected by RNAse protection assay. The liver contained the greatest concentration of siRNA (˜8-10 μg siRNA/g tissue); the spleen, heart and kidney contained lesser amounts of siRNA (˜2-7 μg siRNA/g tissue); and fat tissue contained the least amount of siRNA (<˜1 μg siRNA/g tissue).

Glucose-6-Phosphatase siRNA Detection by RNAse Protection Assay

Balbc mice were injected with U/U, 3′C/U, or 3′ C/3′ C siRNA (4 mg/kg) targeting glucose-6-phosphatase (G6Pase) (see Table 18). Administration was by hydrodynamic tail vein injection (hd) or non-hydrodynamic tail vein injection (iv), and siRNA was subsequently detected in the liver by RNAse protection assay.

TABLE 18 Test iRNA agents targeting glucose-6-phosphatase siRNA Description U/U No cholesterol; dinucleotide 3′ overhangs on sense and antisense strands 3′C/U dinucleotide 3′ overhangs on sense and antisense strands; cholesterol conjugated to 3′ end of sense strand (mono- conjugate) 3′C/3′C dinucleotide 3′ overhangs on sense and antisense strands; cholesterol conjugated to 3′ end of both sense and anti- sense strands (bis-conjugate)

Unconjugated siRNA (U/U) delivered by hd was detected by 15 min. post-injection (the earliest determined time-point) and was still detectable in the liver 18 hours post-injection.

Delivery by normal iv administration resulted in the greatest concentration of 3′C/3′C siRNA (the bis-cholesterol-conjugate) in the liver 1 hour post injection (as compared to the mono-cholesterol-conjugate 3′C/3′U siRNA). At 18 hours post injection, 3′C/3′C siRNAs and 3′C/U siRNA were still detectable in the liver with the bis-conjugate at higher levels compared to the mono-conjugate.

While this invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. 

What is claimed is:
 1. An iRNA agent comprising a sense strand and an antisense strand, wherein the antisense strand and/or the sense strand comprise two or more different modifications, wherein at least one modification is an L sugar modification, and the other modifications are selected from the following group: (a) backbone; (b) 2′-O alkyl; (c) 2′-5′ linkages (d) LNAs, HNAs and/or CeNAs; (e) nucleobase; and (f) cationic groups and/or Zwitterionic groups; wherein the modifications occur 1-6 nucleotides from either end of the strand, and wherein the first four terminal pairs at the 5′ end of the antisense comprises at least two A:U groups.
 2. The iRNA agent of claim 1, wherein the L sugar modification is a L-ribose or L-arabinose modification.
 3. The iRNA agent of claim 2, wherein the L-ribose and L-arabinose modifications further comprise a 2′-H, a 2′-OH or a 2′-OMe.
 4. The iRNA agent of claim 2, wherein at least one of the modifications occurs 1-3 nucleotides from the 3′-end of the antisense strand or the sense strand, or 1-3 nucleotides from either end of the sense strand.
 5. The iRNA agent of claim 1, wherein at least one of the modifications is a 2′-5′ linkage, and wherein the 2′-5′ linkage occurs 1-3 nucleotides from the 3′-end of the antisense strand or the sense strand, or 1-3 nucleotides from either end of the sense strand.
 6. The iRNA agent of claim 1, wherein at least one of the modifications is a 2′-O alkyl modification, and wherein the 2′-O alkyl modification occurs 1-6 nucleotides from the 3′ end and/or the 5′-end of the antisense strand and/or the sense strand.
 7. The iRNA agent of claim 6, wherein the 2′-O alkyl modification is a 2′-OMe modification.
 8. The iRNA agent of claim 1, wherein the iRNA agent is a double stranded iRNA agent comprising a duplex portion of 15-30 nucleotide pairs in length.
 9. The iRNA agent of claim 8, wherein the iRNA agent comprises at least one 3′ overhang of 1-5 nucleotides in length.
 10. An iRNA agent comprising a sense strand and an antisense strand, wherein the antisense strand and/or the sense strand comprises at least one 2′-O alkyl modification and at least one L sugar modification, and optionally one or more 2′-5′ linkages modifications.
 11. The iRNA agent of claim 10, wherein the modifications occur 1-6 nucleotides from either end of the antisense strand or the sense strand.
 12. The iRNA agent of claim 10, wherein the L sugar modification is an L-ribose or L-arabinose modification.
 13. The iRNA agent of claim 12, wherein the L-ribose and L-arabinose modifications comprise a 2′-H, a 2′-OH or a 2′-OMe.
 14. The iRNA agent of claim 12, wherein the modifications occur at the 3′-end of the antisense strand and/or the sense strand.
 15. The iRNA agent of claim 11, wherein the iRNA agent comprises at least one 2′-OMe modification, at least one L sugar modification, and at least one 2′-5′ linkage modification.
 16. A method of increasing nuclease resistance of a duplex comprising an antisense strand and a sense strand, by incorporating two or more different modifications positioned 1-6 nucleotides from either end of the antisense strand and/or the sense strands, wherein at least one modification is an L sugar modification, and the other modifications are selected from the following group: (a) backbone; (b) 2′-O alkyl; (c) 2′-5′ linkages (d) LNAs, HNAs and/or CeNAs; (e) nucleobase; and (f) cationic groups and/or Zwitterionic groups; wherein the first four terminal pairs at the 5′ end of the antisense comprise at least two A:U groups.
 17. The method of claim 16, wherein the L sugar modification comprises a 2′-H, a 2′-OH or a 2′-OMe.
 18. The method of claim 17, wherein at least one of the modifications occurs 1-3 nucleotides from the 3′-end of the antisense strand or the sense strand, or 1-3 nucleotides from either end of the sense strand.
 19. The method of claim 16, wherein at least one of the modifications is a 2′-5′ linkage, and wherein the 2′-5′ linkage occurs 1-3 nucleotides from the 3′-end of the antisense strand or the sense strand, or 1-3 nucleotides from either end of the sense strand.
 20. The method of claim 16, wherein at least one of the modifications is a 2′-O alkyl modification, and wherein the 2′-O alkyl modification occurs 1-6 nucleotides from the 3′ end and/or the 5′-end of the antisense strand and/or the sense strand.
 21. The method of claim 16, wherein the iRNA agent is a double stranded iRNA agent comprising a duplex portion of 15-30 nucleotide pairs in length.
 22. The iRNA agent of claim 21, wherein the iRNA agent comprises at least one 3′ overhang of 1-5 nucleotides in length.
 23. A method of increasing nuclease resistance of a duplex by incorporating at least one 2′-O alkyl modification and at least one L sugar modification and optionally one or more 2′-5′ linkages to an iRNA agent, wherein the modifications occur at the 3′ end and/or the 5′ end of either strand or 1-6 nucleotides from either end of the strand.
 24. The method of claim 23, wherein the L sugar modification is an L-ribose or L-arabinose modification.
 25. The method of claim 23, wherein the L-ribose and L-arabinose modifications further comprise a 2′-H, a 2′-OH or a 2′-OMe.
 26. The method of claim 25, wherein at least one modification occurs at the 3′-end of the antisense strand and/or the sense strand. 